The H-2Kb-restricted cytotoxic T lymphocyte (CTL) response directed against ovalbumin (OVA) is specific for a region contained within the sequence OVA253-276. In this study we have characterized this response by examining the class I-restricted presentation of OVA peptides by the naturally occurring Kb mutant (Kbm) glycoproteins Kbm1, Kbm3, Kbm5, Kbm8, Kbm10, Kbm11 and Kbm23. To facilitate this study we derived a series of somatic cell hybrid targets expressing the various Kbm class I molecules. Experiments using bulk OVA-specific CTL from C57BL/6 mice demonstrated that all the Kbm molecules except for Kbm1 and Kbm8 could present OVA peptides for effective T cell recognition. Clonal analysis revealed a more complex and relatively diverse pattern of CTL recognition of the Kbm/peptide combinations. This diversity is unlikely to result from the existence of multiple, independent Kb-restricted T cell determinants within OVA, since all CTL tested were specific for a single region between residues 259 and 273. Examination of the fine specificity of Kbm presentation identified individual changes at residues 77, 80 and 116 which affected T cell recognition. The results imply that these changes do not inhibit peptide binding since some clones could recognize peptide presented by a particular Kbm molecule, while other clones could not. All three residues reside within the peptide-binding cleft of the class I protein and are not expected to directly contact the T cell receptor. Although we did not formally demonstrate that OVA binding by Kbm vs. Kb is quantitatively identical, our results are best explained by postulating that the changes at residues 77, 80 and 116 indirectly affect T cell recognition by altering peptide conformation. Taken together our results suggest that changes within the class I binding site can profoundly modify peptide presentation without significantly inhibiting peptide-class I association.