Hepatitis B virus (HBV) DNA in hepatocellular carcinoma (HCC) and/or cirrhotic livers of Japanese patients in Japan was investigated by molecular hybridization utilizing a 32P-labeled cloned HBV DNA of adr subtype. Among 24 HCC cases, 9 (37.5%) were positive for serum HBsAg and 10 (41.7%), including 2 cases which were negative for serum HBV markers, were positive for tissue HBsAg in noncancerous portions of the liver. In the latter 10 cases, integrated HBV DNA was detected in HCC. The restriction pattern of integrated viral DNA was different from one case to another. In the noncancerous portion of these 10 HCC cases, free (3/10), random integration (2/10), or clonal integration (1/10) of viral DNA was demonstrated; no hybridization was detected in the remaining 14 HCC cases, either in cancerous or noncancerous portions. Of 6 cases of liver cirrhosis, 2 were positive for serum and histochemical HBsAg; viral DNA was in free state. Viral DNA was not detected in pancreatic tissue in 5 HBsAg-positive cases. The present results are in agreement with previous reports indicating a high frequency of integrated viral DNA in HBV-associated HCC and heterogeneity in the mode of integration. Our results differ in finding no HBV DNA in HCC cases not associated with HBV markers and in finding clonal populations of hepatocytes with integrated HBV DNA less frequently in the noncancerous portion of the liver of HBsAg carriers.