Hepatitis C virus (HCV) infection is currently assessed by detection of antibodies to HCV with immunoassays. However, in the absence of an in vitro system to isolate the virus, or an immunoassay to identify HCV antigen in blood, an ongoing acute or chronic HCV infection can be diagnosed only by detection of HCV RNA by polymerase chain reaction. We used a reverse transcription-nested polymerase chain reaction to detect an HCV 5' noncoding viral RNA sequence in serum specimens collected from anti-HCV-positive individuals belonging to different risk groups and compared the results with those obtained with a prototype recombinant immunoblot assay (Chiron HCV SIA prototype recombinant immunoblot assay [RIBA]) containing four different viral peptides (c22, c33c, c100, and NS5). The prevalence of HCV viremia ranged from 25.9% in HCV antibody-positive blood donors to 92% in HCV antibody-positive hemophiliacs. Elevated alanine aminotransferase values in HCV antibody-positive patients were clearly associated with viremia. Ninety-six percent of HCV RNA-positive patients reacted to two viral antigens or more, compared with only 64% of HCV RNA-negative patients. Contrary to previous reports, HCV viremia was not associated with either the presence or the absence of a particular antibody specificity. The newly introduced NS5 peptide did not improve the sensitivity or specificity of the RIBA. Although 20% of the patients in our study whose sera reacted to all of the antigens were HCV RNA negative, the positive predictive value of a RIBA considered positive by the manufacturer (two or more bands), was rather high (78%) and may allow suspicion of viremia in EIA2 enzyme-linked immunosorbent assay-positive patients.