To investigate the relationship between hepatocarcinogenesis and integration of hepatitis B virus (HBV) DNA in the cellular DNA of the liver, we studied the integration of HBV DNA in various noncancerous regions of the liver from 31 patients using Southern blot analysis. Of 13 patients without hepatocellular carcinoma (HCC), 4 had heterogeneously integrated HBV DNA. Of the latter four patients, two had chronic liver disease, and two had nonspecific histological changes. In contrast, integration of HBV DNA was found in noncancerous tissue from 11 of 18 patients with HCC. In eight patients, homogeneous integration was found in noncancerous tissue, and restriction fragments of integrated HBV DNA were different from those found in cancerous tissue. Moreover, integration of HBV DNA was found in all portions examined from the same liver, and homogeneously integrated HBV DNA showed different restriction patterns in different areas. These results suggest that integration of HBV DNA may occur in heterogeneous sites of cellular DNA before hepatocarcinogenesis. Subsequently, multi-focal clonal populations develop from these hepatocytes, especially frequently in the case of HCC. Integrated HBV DNA may play an important role in the clonal growth of hepatocytes, although the development of HCC requires additional factors.