Priming C57BL/6 mice with dominant antigenic peptides of ovalbumin (OVA) or bovine insulin (INS) in complete Freund's adjuvant generates antigen-specific, H-2Kb-restricted, CD8+ CTL. OVA-CTL produced type 1 cytokines IFN-γ and TNF-α, whereas INS-CTL produced IL-5 and IL-10 with low levels of IL-4 and IFN-γ. Here, we investigate whether differential binding affinities of the OVA and INS peptides to H-2Kb influence the phenotype of the CD8+ CTL. OVA257-264 was found to have significantly higher binding affinity than the INS A-chain12-21 toward Kb. Exchanging the MHC anchor residues between the OVA and INS peptides reversed the Kb binding capacity of the altered peptides. The lower affinity, altered OVA peptides induced CTL that produced IL-5 and IL-10 in addition to IFN-γ, whereas high binding affinity, altered INS peptides induced CTL that produced IFN-γ but not IL-5 or IL-10. These data suggest that MHC binding affinity of peptides can regulate the phenotype of the resulting CD8+ T cells. © 2001 Elsevier Science (USA).