Several serologic studies suggest that infection by hepatitis C virus (HCV) may be associated with the development of hepatocellular carcinoma (HCC). Therefore, we examined tumor tissue and/or the surrounding liver of 20 patients for viral sequences by the polymerase chain reaction (PCR). In 12 cases, liver and tumor tissues were separable for extraction. RNA was extracted from frozen tissues and used as a template for reverse transcription followed by double PCR with nested primers for the 5'-untranslated (NT) and nonstructural NS3 regions of HCV. In addition, the tissue extracts were tested by single PCR for X gene and S gene sequences of hepatitis B virus (HBV). NT region sequences of HCV were detected in the available tumor tissue of all anti-HCV-positive patients except for one. Negative (replicative) strands of HCV RNA were found in the same tissues as positive (genomic) strands at almost the same relative amounts, suggesting replication of HCV in the tumor tissue rather than contamination by HCV-positive blood. HBV X and S sequences were demonstrated in two tumors, but were absent from three tumors that were surrounded by liver tissues with HBV X sequences. One patient had nucleic acids of both viruses in tumor tissue. These observations suggest that in addition to HBV, HCV may play a role in the development of hepatocellular carcinoma.