The seroprevalence of antibodies to hepatitis C virus (HCV) by a second-generation recombinant immunoblot assay (RIBA-2) was tested using 4 recombinant antigens. The results were correlated with those of C100-3 enzyme-linked immunosorbent assay (ELISA) and with the detection of HCV-RNA sequences by the polymerase chain reaction. Sera were obtained from 27 C100-3 ELISA-positive Japanese patients with chronic non-A, non-B liver disease and from 29 C100-3 ELISA-negative patients. All C100-3 ELISA-positive patients and 19 (66%) out of 29 C100-3 ELISA-negative patients reacted to two or more RIBA-2 antigens (reactive by RIBA-2). Of the remaining 10 C100-3 ELISA-negative patients, one patient reacted to just one antigen (indeterminate by RIBA-2), while 9 reacted to none of the 4 antigens (non-reactive by RIBA-2). Forty-three (93%) of the 46 RIBA-2-reactive patients and the single RIBA-2-indeterminate patient tested positive for HCV-RNA sequences, whereas only one (11%) of the 9 RIBA-2-non-reactive patients tested positive. These findings indicate that HCV infection is more prevalent than expected from the results of C100-3 ELISA, and that RIBA-2 is a more sensitive assay for estimating the presence of HCV infection in chronic liver disease.