Gefitinib- or erlotinib-resistant sublines were established by exposing the parental PC-9 cell line to chronic, repeated treatments with these drugs. These resistant sublines showed more than 100-fold more resistance to gefitinib and erlotinib and acquired cross-resistance to other EGFR-TKIs. The T790M EGFR mutation was found by pyrosequencing, and this seemed to be the cause of drug resistance.