A possible causative role for the recently discovered hepatitis C virus (HCV) in the development of hepatocellular carcinoma (HCC) was investigated by assay of sera from HCC patients in Japan for antibodies to a recombinant HCV antigen and to hepatitis B virus (HBV) antigens. Among the 253 HCC patients examined, 156 (61.7%) had no serum markers of either a previous or a current HBV infection (group I), 46 (18.2%) were negative for HBV surface antigen but positive for anti-HBV surface and/or anti-HBV core antibody, indicating the occurrence of a previous, transient HBV infection (group II), and 51 (20.2%) were chronically infected HBV carriers as evidenced by positivity for HBV surface antigen (group III). The prevalence of HCV antibody in group I (68.6%) and II (58.7%) patients was significantly higher than for group III (3.9%) or in 148 additional patients with other (non-HCC) cancers (10.1%) (P less than 0.01). Thus, there appears to be a strong association between HCV infection and the development of HCC, particularly in patients for which HBV infection cannot be implicated as a causative factor. The data also suggest an additional mode of transmission for HCV other than blood transfusion, since a history of blood transfusion was shown in only about 30% of the HCV antibody-positive HCC patients in groups I and II. A high prevalence of HCV antibody was also shown among patients with HCC whose disease was originally thought to be due to very high ethanol consumption.