Integration of hepatitis B virus (HBV) DNA into genomic DNA was investigated in 34 livers bearing hepatocellular carcinoma (HCC) by Southern blot hybridization using 32P-labeled, cloned and purified HBV DNA as a probe. Rehybridization of nitrocellulose paper with a probe containing only the cloning vector was performed after dehybridization to avoid possible false-positive results. Integrated HBV DNA was detected in all 9 hepatitis B surface antigen (HBsAg)-seropositive cases and 3 out of 25 (12%) HBsAg seronegative cases. The hybridization patterns of viral DNA were the same among several cancer nodules in two HCC cases with multiple liver tumors, indicating unicentric hepatocarcinogenesis in these two cases. These results, obtained with avoidance of false-positive results, showed that only a minority of HBsAg-seronegative HCC cases in Japan had demonstrable HBV DNA in the tumors studied by the Southern blot hybridization technique.