To investigate the frequency of exposure to hepatitis C virus (HCV) in chronic liver disease, sera from Japanese patients were tested with the original anti-HCV assay (Ortho) and an anti-HCV assay based on synthetic peptides corresponding to a variety of regions in the HCV genome. Thirty-one (67%) of 46 patients with chronic non-A,non-B hepatitis were anti-HCV-positive by the Ortho ELISA, 20 of whom were also positive by ELISA based on synthetic HCV peptides. Eight (53%) of the 15 patients negative by the Ortho ELISA tested positive for anti-HCV by ELISA based on HCV peptides. Serum HCV RNA was detected in all cases positive for antibody to the HCV peptide and in 14 (78%) of 18 cases without antibody. Thirty-seven hepatitis B virus carriers were without anti-HCV by the Ortho ELISA and were negative for serum HCV RNA, six (16%) of whom were positive by ELISA based on HCV peptides. Antibody responses were directed against each synthetic HCV peptide used, with a considerable difference in incidence, indicating possible expression of the corresponding region in the course of HCV propagation. These findings indicate that exposure to HCV may be more common than expected based on the results of the Ortho ELISA.