A new marker useful for differential diagnosis of cholangiocarcinoma (CC) from hepatocellular carcinoma (HCC) has been identified and investigated. cDNA clone for S100A6 (calcyclin) was identified as a upregulated gene in intrahepatic tumors by differential dot hybridization and Northern blot analysis using normal and tumor intrahepatic tissues. Significant increase in the expression level of S100A6 mRNA was recognized in intrahepatic tissues in three of three CC cases but none of six primary HCC. Immunohistochemical examination of the tumor tissues using monoclonal antibody specific to S100A6 showed that 14 of 18 (77.8%) CC tissue samples were positively stained but only two of 20 (10%) specimens from the patients with primary HCC were weakly stained. These results suggest that the levels of S100A6 mRNA expression and protein in the tissue specimens examined by Northern blot and immunohistochemical staining may be additional useful marker for the differentiation of CC from HCC.