To clarify the role of hepatitis C virus (HCV) infection in chronic liver disease, sera from Japanese patients which were negative by the original anti-HCV assay (Ortho) were subjected to a second-generation anti-HCV assay based on a combination of structural (C22) and nonstructural (C200) recombinant HCV proteins. Of 29 patients with chronic non-A, non-B hepatitis, 20 (69%) were anti-HCV-positive by the second-generation enzyme-linked immunosorbent assay (ELISA) and also positive by the reverse transcription-polymerase chain reaction (RT-PCR) which detects the HCV genome. Of 41 chronic hepatitis B virus carriers, 3 (7%) were positive by the second-generation ELISA; 1 of 3 was positive by RT-PCR. The HCV genome was detected in all cases positive for anti-HCV with high titers. Of 59 healthy subjects negative by the second-generation ELISA, none were positive by RT-PCR. These findings indicate that HCV is a major causative agent of chronic non-A, non-B hepatitis in Japan and that second-generation ELISA is specific and a more sensitive diagnostic assay for chronic hepatitis C.