A paramagnetic microparticle (MP) assay for antibody to hepatitis C virus (anti-HCV) was developed, in which the probe for antibody consisted of synthetic peptides corresponding to HCV capsid and nonstructural c-100 regions, as well as a recombinant protein corresponding to the nonstructural c33c region. Assay performance was evaluated by testing serum from 108 geographically diverse patients with non-A, non-B hepatitis (NANBH). The frequency of anti-HCV reactivity detected with the MP assay and with an enzyme-linked immunosorbent assay (ELISA) for c-100 was 91 and 70 percent, respectively. All c-100 HCV ELISA-reactive specimens also reacted on the MP assay. In addition, anti-HCV seroconversion in three plasma donors was detected one to two blood collection dates earlier by the MP assay than by the c-100 HCV ELISA and at similar blood collection dates by the MP assay and a second-generation anti-HCV ELISA. Serologic responses to the three distinct antigenic regions of HCV in NANBH patients varied: reactivity to all three antigens was most common (49%), reactivity to both capsid and c33c (40%) was next most common, and single-antigen reactivity was rare (4%). MP assay reactivity of 825 volunteer donors was 0.1 percent. These results demonstrate both the utility of additional HCV antigens for an effective anti-HCV screening assay and the application of paramagnetic MP technology to serologic testing for HCV infection.