The immunogenicity and antigenicity of an enzymatically functional (ATPase/helicase) recombinant protein encompassing residues 1207-1612 of the hepatitis C virus (HCV) non-structural 3 (NS3) protein was characterized using B10 congenic mice. Previous studies have indicated a high frequency of NS3-specific antibodies in HCV-infected humans. Similarly, all six immunized murine haplotypes were antibody responders to the NS3 ATPase/helicase domain, with the H-2k and H-2s haplotypes as high responders. As also observed in HCV-infected humans, the murine NS3 antibodies were predominantly directed to conformational determinants. Irrespective of the murine haplotype, IgG1 predominated in the primary anti-NS3 response, whereas IgG1 and IgG2b predominated in the secondary response. The antibody responder hierarchy was reiterated at the T cell level, with the H-2k and the H-2s haplotypes as the best responders. In both the H-2d and H-2k haplotypes ATPase/helicase-primed T cells secreted interleukin 2 and interferon gamma, corroborating observations from HCV-infected humans. In the H-2d, H-2k and H-2s haplotypes the fine specificity of the T cell recognition of the ATPase/helicase domain was further characterized. Multiple, although generally weak, T cell recognition sites were found for all three haplotypes. The large size of the NS3 protein together with the presence of multiple class II binding motifs explain the high prevalence of NS3 antibodies in immunized mice and predict a similar explanation for the observed high frequency of NS3-specific antibodies in HCV-infected humans.