Hepatocellular carcinoma tissues from HBsAg-negative patients with chronic alcoholic liver disease were investigated for the presence of hepatitis B virus DNA. Southern blot analyses of DNA extracted from the hepatocellular carcinomas were negative for hepatitis B virus DNA in all 17 patients examined, at a level of sensitivity of less than 0.01 genome equivalent per cell. Similarly, in liver tissues from another 30 patients with alcoholic cirrhosis without hepatocellular carcinoma, no hepatitis B virus DNA was detectable. We conclude that in our patients there is no molecular evidence for a contribution of hepatitis B virus infection to the development of hepatocellular carcinoma in alcoholic liver disease.