Quantitative immunohistochemistry of ERBB-2 and MET receptor proteins and of cyclooxygenase 2 (COX-2) was undertaken to determine if there is a positive correlation between overexpression of either ERBB-2 or MET and up-regulation of COX-2 in human cholangiocarcinogenesis. ERBB-2, MET, and COX-2 immunoreactivities were measured in cancerous parenchyma of 71 archival cases of human cholangiocarcinoma (ChC) compared with hyperplastic small biliary ducts in surrounding nonneoplastic liver and with bile ducts of normal adult human liver. ERBB-2, MET, and COX-2 immunoreactivities were also assessed in both large and small hyperplastic biliary ducts (HBDs) in 27 archival cases of hepatolithiasis and 20 archival cases of primary sclerosing cholangitis (PSC), both of which are risk conditions for human cholangiocarcinogenesis. There was a strong positive correlation between increased ERBB-2, but not MET, and COX-2 immunoreactivity measured in the tumors and risk conditions. Enhanced immunoreactivity for ERBB-2 and COX-2 also correlated directly with tumor differentiation and was highest in well-differentiated tumors. Interestingly, some but not all cases of hepatolithiasis and most cases of PSC showed increased ERBB-2 and COX-2 immunostaining in the large but not small HBDs, whereas strong MET immunostaining was detected in both the large and small ducts. In conclusion, overexpression of ERBB-2 and COX-2 may herald an early carcinogenic event in the human hepatic biliary tract and one that is consistent with a frequent anatomic site of origin of the tumors. The results also suggest ERBB-2 and COX-2 as potentially important targets relevant to chemoprevention or adjunct therapy of ChC.