The authors successfully established 5 hepatocellular carcinoma cell lines, JHH-1, 2, 4, 5 and 6, derived from hepatitis B surface antigen seronegative patients and one line. JHH-7, from a patient considered to be a hepatitis B virus carrier. In the culture media of any of the JHH cell lines, including JHH-7, hepatitis B surface antigen was not detected by radioimmunoassay. However, in JHH-7, integration of hepatitis B virus DNA was confirmed at two sites on the chromosomes of this line by Southern blot hybridization. In contrast, in other JHH cell lines derived from hepatitis B surface antigen seronegative patients, integration of hepatitis B virus DNA into the chromosomes of cells was not detected. These cell lines will be useful in the investigation of hepatocellular carcinoma development, especially for research into non-A/non-B hepatitis viruses.