Serum samples from 87 patients with hepatocellular carcinoma (HCC) in the United States were tested for evidence of hepatitis C viral (HCV) infection using an immunoblot assay for antibodies to the hepatitis C virus and the polymerase chain reaction to detect HCV RNA. The findings with these assays were compared to those with a first generation enzyme-linked immunoassay (EIA). Antibody to HCV (anti-HCV) was detected in 14 patients (16%) by EIA; only eight of these were also positive by immunoblot and four had HCV RNA by reverse transcription polymerase chain reaction (RT-PCR). An additional four cases, negative by EIA, were found to be positive by immunoblot; two of these had HCV RNA in serum. Evidence of previous hepatitis B viral infection was noted in 15 patients (17%). Only two patients with antibody to hepatitis B core antigen also had anti-HCV by the immunoblot assay, suggesting that concomitant infection with the hepatitis B and C viruses was not common. Thus, HCV infection appears to play a less important role in the pathogenesis of HCC in the United States than in southern Europe and Japan and other etiologic factors should be sought in this population.