Bile acids are implicated as aetiological factors in many types of gastrointestinal tract cancer including cholangiocarcinoma (CCA). Alterations in bile acid concentrations may affect the pathogenesis of these different types of cancer. Our aim was to determine the bile acid profile in gallbladder bile from patients who underwent liver resection. Thirty-seven patients with cholangiocarcinoma, 5 with hepatocellular carcinoma, and 7 with benign biliary diseases were studied. High pressure liquid chromatography was used to analyze conjugated and unconjugated bile acids. CCA patients with low (≤ 2 mg/dl) and high (>2 mg/dl) levels of total serum bilirubin had significantly higher total bile acid and conjugated bile acid concentrations than the benign biliary disease group. Markedly elevated levels of cholic and chenodeoxycholic acid were found in CCA cases with high levels of total serum bilirubin. Concentrations of total bile acids and primary bile acid were correlated with serum cholesterol, bilirubin and ALP in CCA. Notably, correlation of the carcinoembryonic antigen, a tumor marker, was found with level of total bile acids and chenodeoxycholic acid. These findings suggest a different pattern of bile acid concentration in cancer patients compared to patients with benign biliary diseases. Thus, accumulation of certain bile acids may be involved in carcinogenesis.