The prevalence of anti-hepatitis C virus (HCV) enzyme immunoassay (EIA)-positive in 167,511 Australian volunteer blood donors from Adelaide, Melbourne, Perth, and Sydney was 0.78%. One thousand two-hundred and eighteen EIA-positive serum samples were assessed by supplemental tests including a blocking EIA and two peptide EIAs corresponding to major epitopes of the HCV C-100-3 antigen. Seven hundred and eighteen samples (59%) were negative by supplemental testing; no evidence of reactivity with other HCV gene products or HCV RNA detected by cDNA polymerase chain reaction was found in selected samples from this group. In contrast, of 43 samples randomly selected from 400 samples (32.8%) positive by supplemental testing, 88% were reactive with HCV 33-C or core antigens and 52% contained HCV RNA, suggesting contact with HCV and infectivity of most donors in this group. Most samples equivocal by supplemental testing reacted only with C-100 and not with other HCV antigens when tested by dot immunoblot assay. Only 21% had detectable HCV RNA. The battery of assays used in this study indicated that approximately 32% of HCV EIA repeatably reactive serum samples were serologically related to HCV, corresponding to a 0.25% prevalence of potentially infectious donors.