Endoscopic biopsy has limited accuracy in diagnosis of ampullary tumors. Endoscopic biopsy specimens and surgically resected specimens in a collective series of 78 Japanese patients with ampullary tumor were retrospectively reviewed to investigate the clinical implications of endoscopic biopsy. Endoscopic biopsy specimens were classified into five groups based on the degree of epithelial atypia: group 1 (no atypia), group 2 (mild atypia), group 3 (moderate atypia or adenoma), group 4 (severe atypia or carcinoma in situ), and group 5 (invasive carcinoma). Final diagnosis of the 78 resected ampullary tumors was adenoma in five cases, carcinoma in 27 cases, and both adenoma and carcinoma in 46 cases. Biopsy accuracy of carcinoma (group 4 or 5) was 70% (51 of 73) overall in 73 carcinoma cases. Biopsy accuracy was 50% (7 of 14) in the intramural protruding type, 64% (21 of 33) in the exposed protruding type, and 88% (23 of 26) in the ulcerating type. The diagnostic accuracy of adenoma (group 3) was 80% (4 of 5) in five cases of ampullary adenoma. In 18 (25%) of the 73 carcinoma cases, biopsy diagnosis was adenoma (group 3), whereas carcinoma was found in the deeper layers of surgically resected specimens. Biopsy diagnosis of adenoma does not rule out the possibility of deeper carcinoma in ampullary tumors. 