The purpose of this study was to compare the results of supplementary testing of volunteer whole blood donors who had been screened by the first hepatitis C virus antibody assay licensed in the United States with results from donors screened by a newer, more sensitive, multi- antigen assay. In contrast to the earlier assay, the multi-antigen assay incorporates a recombinant hepatitis C virus antigen, c22-3, which is encoded by a structural region of the viral genome. Supplementary testing included a second-generation recombinant immunoblot assay and a highly sensitive polymerase chain reaction assay for evidence of hepatitis C virus genomic RNA. A comparison of supplementary test results reveals a higher percentage of donors screened by the newer assay to be indeterminate on recombinant immunoblot (34.4% vs. 6.4%, p < 0.05). Furthermore, polymerase chain reaction testing of donors with indeterminate blot results shows that 14 percent have evidence of viral RNA. For this reason, counseling of donors with indeterminate patterns on immunoblot must include informing them of the possibility that they are infected.