Recent studies suggest that Epstein-Barr virus (EBV) may act as a helper virus for the development of hepatocellular carcinoma by promoting replication of the hepatitis C virus (HCV) in the infected liver. Detection of EBV DNA in a high percentage of HCV-positive human hepatocellular carcinomas (HCC) from Japanese patients has supported this concept. In order to determine whether EBV infection is associated with HCC, we examined paraffin-embedded tissues from 31 cases of non-cirrhotic livers with hepatocellular carcinoma for the presence of EBV, HCV and hepatitis B virus (HBV) infection. RNA prepared from tumor samples were used as a template for reverse transcription followed by double-nested PCR with primers for the 5' untranslated region (NT) of HCV. DNA extracts of tumor samples were tested by single polymerase chain reaction for the detection of EBV and HBV (X- and/or S-gene) DNA sequences. To control for nucleic acid integrity, all tumor samples were amplified for human beta-globin DNA by polymerase chain reaction and subjected to Southern blot hybridization. None of the cases was found to be positive for EBV. Ten HCC cases (32%) tested positive for HCV and 12 HCC cases (38%) tested positive for HBV. Six of the surveyed patients had nucleic acids of both HCV and HBV in their tumor tissue. All HCC tumor samples were positive for beta-globin. Our study shows that HCV and HBV infections, but not EBV infection, are associated with hepatocarcinogenesis in non-cirrhotic livers. Other unknown risk factors seem to be in effect in the development of hepatocellular carcinoma in non-cirrhotic livers.