Aberrations of the PTEN, Smad4 and DCC genes have not been determined in biliary tract cancers. We performed allelotype analysis to screen for alterations of these genes. We looked for the presence of allelic imbalance (AI) at the PTEN and Smad4 genes in extrahepatic bile duct (EHBD) and ampullary cancers using polymorphic microsatellite markers. These tumors were also examined for AI at the DCC gene using polymerase chain reaction amplification of variable numbers of tandem repeats. AI at the PTEN, Smad4 and DCC genes was observed in 5.3%, 8.3% and 20.7%, respectively, of EHBD tumor cases. AI at the PTEN, Smad4 and DCC genes was detected in 13.3%, 50% and 8.3%, respectively, of ampullary cancer cases. Our results suggest that (a) alteration of the Smad4 gene is a major factor in the development of ampullary cancer and (b) PTEN, Smad4 and DCC genes are altered infrequently in EHBD cancers.