In order to clarify the relationship between the integration of hepatitis B virus (HBV) DNA and human hepatocellular carcinoma (HCC), the states of HBV DNA in liver tissues were examined by the Southern blot hybridization procedure. Integration of HBV DNA was found in 12 of 25 HCC cases and 5 of 12 cirrhotic cases. Of these 17 cases, 11 were positive for serum hepatitis B virus surface antigen (HBsAg) and the remaining six were positive for more than one of the serum HBV-related antibodies. In all three cases of chronic hepatitis and 18 controls, integration of HBV DNA could not be detected. Free viral DNA was found in 12 of 15 cases with serum HBsAg. One patient without serum HBsAg also had free viral DNA in the liver. Integration of HBV DNA could be observed in HCC cases positive for serum HBsAg at the highest frequency. However, there was one HCC case from an HBsAg carrier in whom integration of HBV DNA might not have a causal effect on malignant transformation, because integrated HBV DNA could be detected only in a non-tumor region. Since integrated HBV DNA could not be detected in 13 of 25 HCC cases, other etiologic factors than the integration of HBV DNA must also be taken into consideration for HCC.