Polymerase chain reaction was used to investigate the presence of the hepatitis B and C viruses in liver tissue from Taiwanese patients with hepatocellular carcinoma by examining paired samples (tumor and non-tumor) from 38 cases. We used a DNA-polymerase chain reaction protocol with primers spanning the regions of the hepatitis B virus genome corresponding to HBs, HBc, and HBx genes and RNA-polymerase chain reaction protocol with primers spanning the 5' untranslated region of the hepatitis C virus. Co-infection with hepatitis B and hepatitis C viruses was seen in nine patients (23%), only three of whom had anti-hepatitis C virus in serum. One of these three was HBsAg-negative in serum while the other two and four of the other six from this group were HBsAg-positive. One of the patients with anti-HCV and no HBsAg in serum had no hepatitis C virus-RNA in liver tissue, while hepatitis B virus-DNA was detectable by using the HBc and HBx specific primers. We detected hepatitis C virus as a single agent in the liver in only one patient. This patient was anti-HCV positive and HBsAg-negative. The remaining 27 patients (71%) had infection with hepatitis B virus only. Twenty-five of 27 patients had HBsAg in their sera. HBs-specific primers detected hepatitis B virus-DNA in non-tumor tissue from 23 patients and in tumor tissue from 25 patients. HBc-specific primers detected hepatitis B virus-DNA in non-tumor tissue from 24 patients and in tumor tissue from 20 patients. Finally, HBx-specific primers detected hepatitis B virus-DNA in non-tumor tissue from 24 patients and in tumor tissue from 25 patients. These data indicate that in a hyperendemic area, hepatitis B virus is closely associated with the development of hepatocellular carcinoma but that infection with hepatitis C virus may play a secondary role.