We studied the frequency of hepatitis B virus replication in Chinese patients with hepatocellular carcinoma. Hepatitis B e antigen and hepatitis B virus DNA could be detected in the sera of 28% and 47% of 116 HBsAg-positive patients, but not in the sera of 15 HBsAg-negative patients. Replicative forms of hepatitis B virus DNA were detected in the neoplastic and nonneoplastic liver tissues from 34% and 62% of 29 HBsAg-positive patients and 0% and 20% of five HBsAg-negative patients by Southern blot hybridization analysis. Of the 10 patients with chronic hepatitis B virus infection in whom hepatocellular carcinoma developed during follow-up, hepatitis B e antigen and hepatitis B virus DNA were detected in the sera of seven and eight patients, respectively, at presentation, 13 to 43 mo before the diagnosis of hepatocellular carcinoma. In nine patients, hepatitis B virus DNA was persistently or intermittently detected in the serum during follow-up. Five patients remained hepatitis B e antigen-positive and seven were detectable for hepatitis B virus DNA in serum when hepatocellular carcinoma was diagnosed. Four patients had one or more episodes of exacerbations before the diagnosis of hepatocellular carcinoma; in three, the exacerbations were associated with changes in level of hepatitis B virus replication. Our study demonstrated that despite the long interval between the onset of hepatitis B virus infection and the development of hepatocellular carcinoma, hepatitis B virus replication persisted in most patients with hepatocellular carcinoma, albeit at a low level.