Serum tumor markers such as alpha-fetoprotein (AFP), carcinoembryonic antigen, carbohydrate antigen (CA) 19-9, CA242, and CA50 were analyzed to evaluate their diagnostic values in single and combined tests for distinguishing intrahepatic cholangiocarcinoma (ICC) from hepatocellular carcinoma (HCC). Preoperative serum levels of AFP, carcinoembryonic antigen, CA19-9, CA242, and CA50 were measured in 45 ICC and 76 HCC patients. The serum levels and the positive rate of AFP, CA19-9, and CA242 were significantly different between the ICC patients and HCC patients. Although AFP (-) was the most sensitive assay for distinguishing ICC from HCC (91.1%), its specificity was significantly lower than that of CA242 (+) and CA19-9 (+). The combination of AFP (-) and CA242 (+) afforded a high specificity of 94.3 per cent and showed highest accuracy (78.5%). Evaluation of patients without liver cirrhosis also showed similar results. The diagnostic value of CA242 (+) is better than that of CA19-9 (+) and AFP (-) in distinguishing ICC from HCC. Combined detection of AFP (-) and CA242 (+) can improve the specificity and accuracy of diagnosing ICC.