To elucidate the possible role of pancreatic juice reflux into the biliary tract in promoting the development of biliary carcinoma, Syrian hamsters were subjected to cholecystoduodenostomy and ligation of the distal end of the common duct and then subcutaneously injected with N-nitrosobis(2-oxopropyl)amine (BOP) (experimental group). The incidences of gallbladder carcinoma and extrahepatic bile duct carcinoma in the experimental group was significantly higher than in the sham-operated group (P < 0.01, P < 0.05). The proliferating cell nuclear antigen (PCNA) labeling indices of both regions gradually increased with time, and were significantly higher in the experimental group at weeks 9 and 16 than in the sham-operated group at the same time. Trypsin and phospholipase A2 (PLA2) activities in bile and tissue levels of superoxide dismutase (SOD) in the gallbladder and extrahepatic bile ducts were higher in the experimental group than in the sham-operated group. These findings suggest that the carcinogenic effect of BOP was enhanced in biliary epithelium that had proliferated in response to and/or had been injured by activated pancreatic enzymes refluxing into the biliary tract and then increased free radical activity, leading to a high frequency of carcinoma development in the biliary tract.