AIM To investigate the hepatitis C virus (HCV) infection in the tissues of carcinoma of extrahepatic bile duct and study their correlation. METHODS HCV NS5 protein and HCV RNA were detected by labeled streptavidin biotin (LSAB) method and in situ reverse transcription polymerase chain reaction (IS-RT-PCR) in sections of 51 cases of carcinoma of extrahepatic bile duct and 34 cases of control group (without malignant biliary disease). RESULTSIn 51 cases of carcinoma of extrahepatic bile duct, HCV NS5 protein was detected in 14 (27.5%), which was clearly stained in the cytoplasm of cancer cell but not in the nucleus or cell membrane. HCV RNA was detected in 18 (35.4%), which was located in the nucleus of cancer cell in 12 cases and in the cytoplasm in 6 cases. HCV NS5 protein and RNA coexistence was found in 2 cases. In 34 cases of control group, HCV RNA was detected in 2 (5.9%). HCV NS5 protein and RNA positive cells were found either scattered or in clusters. CONCLUSION The prevalence of hepatitis C viral infection in the tissues of carcinoma of extrahepatic bile duct was significantly higher than in control group (χ χ χ χ χ 2 =9.808, P=0.002). The findings suggest a correlation between HCV infection and carcinoma of extrahepatic bile duct, which is different from the traditional viewpoint. HCV infection might be involved in the development of carcinoma of extrahepatic bile duct.