Current criteria for a reactive (positive) interpretation on hepatitis C virus (HCV) recombinant immunoblot assay (RIBA) require > or = 1+ reactivity to at least two of the four HCV antigens present in the assay. Given that 5-1-1 is a subcomponent of c100-3, there is concern that donor samples reacting only with these two antigens (and not with c22-3 or c33c) could be incorrectly classified as positive on the basis of limited reactivity to only one HCV gene product. It is determined that 0.23 to 0.44 percent of HCV enzyme immunoassay-repeatably reactive donor sera demonstrate a pattern of 5-1-1 and c100-3 only on RIBA. Evaluation of six such donor sera using peptide enzyme immunoassays spanning the c100-3 antigen showed highly restricted reactivity to the 5-1-1 N-terminal region of c100-3, in contrast to broad 5-1-1 and c100-3 C-terminal peptide reactivity observed in the majority of donor sera with other positive RIBA patterns. HCV polymerase chain reaction and follow-up serologic evaluations of four of these donors indicated the absence of viremia or evolving seroconversion in all cases. It is concluded that, in the blood donor setting, a pattern of only 5-1-1 and c100-3 reactivity is typically not indicative of HCV infection. To avoid overinterpretation, it is recommended that RIBA grading criteria be revised to require reactivity to two or more HCV-encoded gene products.