We investigated mechanistic differences in antigen presentation between murine MHC class I variants H-2K(b) and H-2K(bm)8. H-2K(bm)8 differs from H-2K(b) by four residues at the floor of the peptide-binding site, affecting its B pocket which interacts with the second (P2) residue of the peptide. The rest of the molecule, including the T cell receptor (TCR)-contacting residues, is identical to H-2K(b). Due to this variation, CTLs that recognize the ovalbumin 257-264 and HSV gB 498-505 peptides on H-2K(b) cannot recognize them on H-2K(bm)8. This could be due to impaired peptide binding or an altered peptide: K(bm)8 conformation. Peptide binding studies ruled out the first explanation. Molecular modeling indicated that the most obvious consequence of amino acid variation between peptide/H-2K(b) and peptide/H-2K(bm)8 complexes would be a loss of the conserved hydrogen bond network in the B pocket of the latter. This could cause conformational variation of bound peptides. Intermolecular second-site reversion was used to test this hypothesis: P2-substituted OVA and HSV peptides, engineered to restore the hydrogen bond network of the B pocket, were the only ones which restored CTL recognition. These results provide a molecular understanding of peptide/MHC conformational variation.