We examined expression of α-amylase isozymes (pancreatic and salivary), trypsin and pancreatic lipase on the epithelium of extrahepatic peribiliary glands immunohistochemically using 53 autopsied normal extrahepatic bile ducts. Three parts of the extrahepatic bile duct (common bile duct, intrapancreatic bile duct and bile duct at the ampulla of Vater) were examined in each case. Histologically, the extrahepatic bile duct harbored branched tubular glands (extrahepatic peribiliary glands). Extrahepatic peribiliary glands were few in the common bile duct and intrapancreatic bile duct and numerous in the bile duct at the ampulla of Vater. Immunohistochemically, pancreatic α-amylase was expressed in the epithelium of extrahepatic peribiliary glands in 42 cases (79%). Salivary α-amylase was expressed in the epithelium of the glands in 38 cases (72%). Trypsin was expressed in the epithelium of the glands in 32 cases (60%). Pancreatic lipase was expressed in the epithelium of the glands in 45 cases (85%). The immunoreactivity of these enzymes was granular and located in the supranuclear cytoplasm (corresponding to the Golgi apparatus) of the epithelium of the glands. We confirmed the specificity of the immunoreactivity of these enzymes with various methods. These results suggest that extrahepatic peribiliary glands produce α-amylase isozymes, trypsin and pancreatic lipase and secrete these enzymes into lumens of the extrahepatic bile duct. The secreted enzymes may play an important role in the physiology of the extrahepatic bile duct and bile. (HEPATOLOGY 1993;18:803-808).