Hepatitis derived from hepatitis B virus (HBV) infection is endemic throughout the world, but it is particularly prevalent in Asia and Africa. In these areas, demographic studies show a strong coincidence between HBV infection (assayed by HBV antigenic markers) and the incidence of primary liver cancer. On these grounds, a causal link between HBV infection and primary hepatocellular cancer has been proposed. Recently, a human hepatoma cell line (PLC/PRF/5; Alexander cells) has been shown to produce hepatitis B surface antigen (HBsAg). We show here that the Alexander cell line contains at least six (four complete and two partial) hepatitis B viral genomes integrated into high molecular weight host DNA. An analysis using specific probes to fragments of the HBV genome suggests that integration of the virus in most cases occurs at the nicked cohesive end region of the virus. Expression of viral sequences using Northern blots demonstrates the presence of RNA transcripts specific for the surface antigen sequences of HBV DNA and the absence of detectable transcripts corresponding to the hepatitis B core antigen.