More than 80 Conservative MPs defied his orders and backed a referendum on Britain’s membership of the European Union.

The morning after the vote, Michael Gove hit the airwaves to deny the rebellion would lead to a "convulsion" in the Tory party or that it was a "humiliation" for the Prime Minister.

He insisted the government and MPs were "united" behind a goal to get back powers from Europe.

In Monday's vote a total of 81 of Mr Cameron's MPs voted for a Commons motion calling for a referendum on Britain’s relationship with the EU, even the PM had ordered his party to oppose it. It included two tellers indicated they supported the motion.

Another two Tories voted yes and no, the traditional way of registering an abstention. A further 12 did not vote.

In all, about half of all Conservatives outside the “payroll vote” of ministers and their aides scorned Mr Cameron’s authority, with most of them coming from the 2010 intake of MPs. Rebel leaders warned that the Prime Minister faced a protracted “war” with his own party over the European issue.

The call for a referendum was defeated only because Labour and Liberal Democrat MPs also opposed it. The motion was rejected by 483 votes to 111 in the late-night Commons vote.

At least two junior members of the Government backed the referendum motion and resigned. They included Adam Holloway, an aide to David Lidington, the Europe Minister, who accused ministers of mistreating loyal Conservatives.

Previously, the biggest Conservative rebellion over Europe came in 1993 when 41 Tory MPs defied John Major over the Maastricht treaty. The revolt was also easily the biggest that Mr Cameron’s government has suffered on any issue.

Many Conservative MPs are unhappy about aspects of Mr Cameron’s Coalition deal with the Liberal Democrats, and the new mood of defiance raises the prospect of more challenges to the Prime Minister’s authority in future.

Mr Cameron was warned that Europe would continue to dog his premiership after the record Conservative revolt.

After helping inflict on the Prime Minister the biggest ever Tory rebellion over Europe, senior backbench MP Mark Pritchard insisted it would become "more rather than less of an issue" over the rest of the Parliament.

After the vote, David Nuttall, the Conservative MP who proposed the referendum motion, also warned ministers that they had “won this particular battle but they certainly have not won the war".

Ed Miliband, the Labour leader, described the vote as “a humiliation” for the Prime Minister. “If he can’t win the argument with his own backbenchers, how can the country have confidence that he can win the arguments that matter for Britain?” he said.

Sir George Young, the Leader of the House, denied that the Conservatives were split, saying: “It is not Maastricht.”

Michael Fallon, the Conservative deputy chairman, said the revolt was “not that huge”. “Colleagues feel very strongly about Europe and obviously they feel that they need to let off steam about this particular issue,” he said.

On Tuesday Michael Gove, the Education Secretary, tried to smooth over the divisions, claiming the party was "united as never before" on the need to repatriate powers.

Despite admitting there were a "significant numbers of people who wanted to take a differing view", he denied there were "convulsions" in the party, adding that disagreements had been conducted with "cordiality".

The Prime Minister, he added, brought about the vote because he "speaks from the heart". But he added that the government and MPs were "united" behind a goal to get back powers from Europe.

He told BBC Radio 4's Today programme that while there were a "significant number" of rebels, differring opinion between Cabinet and backbenchers were not significant and could be "exaggerated".

He said the Prime Minister "wants to refashion our relationship with the European Union" and was "committed" to taking back powers to boost economic growth.

"It was a very precisely worded motion which allowed a number of people like myself, who are passionate Eurosceptics, to say: Look, I disagree with the tactics but we agree on the ultimate goal," he said.

"If you have a disagreement about tactics... and if you have that disagreement conducted with cordiality and good manners on all sides then I don't think it leads to the sorts of convulsions that many people want it to.

"It's not a humiliation. What we have is a coalition and because we have a coalition, all parties need to compromise in the national interest."

But pressed repeatedly as to whether issues such as employment regulations could be renegotiated within this Parliament, he said: "I have all sorts of timetables".

He added: "But the critical thing is that when events change, you have got to change your mind as well and events are changing in Europe at a remarkably fast rate."

Pushed again, he said: "I would like to see a change in this Parliament.

"We are already winning powers back - we need to win more and that process will require careful negotiation but what we are fortunate in having is a Conservative Party that is united as never before behind that renegotiation."

Deputy Prime Minister Nick Clegg condemned the backbench rebellion as a "smash-and-grab dawn raid".

During a visit to London's East End to discuss the coalition's stance on regulation with local hi-tech businesses, Mr Clegg said Britain should be leading, not leaving Europe.

Recognising that reforms to the EU were necessary, he said: "You don't change Europe by launching some smash-and-grab dawn raid on Brussels.

"You do it by setting out the case for changes and then arguing the case with other countries.

"We can't do this on our own, we have to build alliances, we have to convince and persuade other countries and that is what we look to do all the time."

One of those Tories who backed the motion, Douglas Carswell, said it was the night Euroscepticism “went mainstream” in the Commons.

“Finally, people in the Westminster bubble are beginning to reflect the real concerns that people out there have about Europe,” he said.

Mr Cameron’s aides insisted that he would emerge from the rebellion with the respect of voters because he had refused to back down.“The Prime Minister is standing firm on his policy. He doesn’t have any regrets,” said a Downing Street source.

A No10 spokesman said after the vote: “The House of Commons has clearly voted against this motion.

“We understand that many people who voted for it felt very strongly – and we respect that. However, the Government has to do what is in the national interest.

“The easy thing to do would have been for us to have avoided expressing a view. It was important to take a strong lead because Britain’s best interests are served by being in the EU.

The PM has made clear that he shares the yearning for fundamental reform of the EU and is determined to achieve that.”

Earlier, during more than six hours of debate, a string of Conservative MPs accused Mr Cameron of mishandling the EU referendum issue by imposing a three-line whip on the vote, the strongest possible order to back the Government.