Purpose Measurement of the activities of alkaline phosphatase isoenzyme has been used for the identification and monitoring of diseases associated with the isoenzyme. Biliary alkaline phosphatase (BALP), an isoform of liver-ALP, has been found in the serum of patients with biliary obstruction and metastatic liver cancer. This study compared the BALP isoform in the serum of patients with cholangiocarcinoma (CCA) with that of non-jaundiced benign hepatobiliary disease, other cancers, and healthy persons. Methods ALP isoforms were separated using cellulose acetate electrophoresis and the activity was demonstrated using indolyl blue reagent. Results The BALP isoform was demonstrated in 65% of CCA patients independently of jaundice condition or histological grading of the tumor. The level of serum BALP in non-jaundiced CCA was significantly lower than that of jaundiced CCA, and not correlated with serum bilirubin. No BALP was detected in healthy persons. In the patients with high serum ALP (>147U/l), BALP can differentiate non-jaundiced CCA patients from other non-jaundiced carcinoma patients with 85% sensitivity, 79% specificity, and positive and negative predictive values of 81% and 83%, respectively. Conclusion The demonstration of serum BALP, in particular in non-jaundiced patients with high serum ALP, may indicate the presence of tumor in the bile duct. [ABSTRACT FROM AUTHOR]