Second generation hepatitis C virus (HCV) Elisas are currently in use in Europe and have been submitted for approval in the United States. These new assays contain additional antigens from the putative nucleocapsid and NS-3 regions of the HCV genome in addition to the c100-3 antigen present in first generation Elisas. A supplementary test, the second generation RIBA (Chiron Co trademark) HCV strip immunoblot assay (2-RIBA HCV SIA) has also been developed. The strip immuno-blot assay uses four recombinant HCV antigens (5-1-1 [NS-4], c100-3 [NS-4], c33c [NS-3], and c22-3 [NS-3 [nucleocapsid]) slot blotted on nitrocellulose. Screening of random volunteer blood donors with the Ortho (Ortho Diagnostic Systems trademark) second generation HCV Elisa (2-Ortho HCV Elisa) indicates that a substantial change in the repeat reactive donor population is observed with the new test. Two notable features of this change are: i) a large number of samples reactive in the 2-RIBA HCV SIA for the second generation antigens, c33c and c22-3, are detected by the 2-Ortho HCV Elisa; ii) the percentage of 2-Ortho HCV Elisa reactive specimens found indeterminate (reactive for only one HCV antigen) by the 2-RIBA HCV SIA is higher than in first generation HCV Elisas (approximately 25 versus 5%). In addition, 2-ortho HCV Elisa repeat reactive, 2-RIBA HCV SIA indeterminate samples are dominated by reactivity to c22-3 instead of c100-3 which is the case for first generation HCV Elisa repeat reactive samples.(ABSTRACT TRUNCATED AT 250 WORDS)