Antibodies against a possible causative agent of non-A, non-B hepatitis, hepatitis C virus (HCV), in Japanese patients with hepatocellular carcinoma were analyzed using the enzyme-linked immunosorbent assay (ELISA) system from Ortho Diagnostic Systems, Japan. Fifty of 58 cases of hepatitis B virus surface antigen (HBsAg)-negative hepatocellular carcinoma were positive for the antibody (86%) and 8 of 42 cases of HBsAg-positive hepatocellular carcinoma were positive (19%). Among patients with HBsAg-negative hepatocellular carcinoma, the prevalence of the antibody was greater among those who had received a blood transfusion (97%) than among those with no history of transfusion (70%). Only 3 of 54 patients with cancers other than hepatocellular carcinoma were found to be antibody-positive (5.6%) and all three patients had a history of blood transfusion. These results show a close relationship between the presence of anti-HCV antibody and HBsAg-negative hepatocellular carcinoma in Japan.