A combination of different enzyme immunoassays (EIAs) was used for the serological confirmation of sera that were positive in a hepatitis C virus (HCV) second-generation screening EIA. Different reaction patterns were related with the probability of the HCV-carrier state as determined by polymerase chain reaction (PCR). Five hundred and eight sera of volunteer blood donors were send for confirmation and at first reexamined with both Abbott and Ortho second-generation screening EIA. A group of 195 sera, positive in both assays, was further evaluated by the Abbott Supplemental Assay, the Monolisa anti-HCV and an EIA with only the amino terminal part of the nucleocapsid protein as antigen. In addition PCR on the 5'-noncoding region of the viral genome was performed. We observed that 75 of the 78 PCR-positive sera were found in a group of 89 sera that were strongly positive in the four EIAs used. Moreover all but 1 PCR-positive sera were reactive against the nucleocapsid protein of the virus. Hence we concluded that a genuine antibody response to the nucleocapsid protein is highly suggestive for the HCV-carrier state.