Mutations in the first and second exons of Ha-, Ki- and N-ras oncogenes were investigated in 17 epithelial tumors of the ampulla of Vater by single-strand conformation polymorphism analysis and direct sequencing of DNA fragments amplified by polymerase chain reaction. The panel included 12 intestinal-type adenocarcinomas, 3 villous adenomas, 1 papillary carcinoma and 1 neuroendocrine carcinoma. Six cases (35%) contained ras mutations, affecting codon 12 of Ki-ras in 2 adenomas and 3 carcinomas, and of N-ras in 1 adenoma. All mutations were found in adenomas and among cancers with adenomatous areas, whereas none of the cases lacking adenomatous areas contained mutations. This suggested that ampullary cancers represent heterogeneous diseases with respect to the presence or absence of adenomatous areas and, among those with adenomatous areas, with respect to the presence of activated ras genes. Ki-ras mutated cases included 3 of 4 tumors which mainly involved the intraduodenal bile duct, thus suggesting that a proportion of Ki-ras-mutated ampullary cancers might correspond to those originating from the epithelium of the bile duct component of the ampulla.