The immunodominant ovalbumin257-264 (OVA-8, SIINFEKL) and herpes simplex virus gB496-503 (HSV-8, SSIEFARL) peptides share 50% amino acid identity (residues P1, P3, P5 and P8) and bind with comparable efficacy to the murine MHC-encoded class I molecule H-2Kb. However, these two peptides bind differently to H-2Kbm8, a natural H-2Kb variant with a substitution in four amino acids on the floor of the peptide-binding site; HSV-8 binds with high and OVA-8 with a relatively low efficacy. To investigate which of the non-homologous peptide residues were responsible for this differential binding, we used substituted peptide variants and the class I thermodynamic stabilization assay. Variation at the solvent-exposed peptide residues P6 and P7 did not appreciably influence binding. By contrast, variation at the buried P2 and, surprisingly, at the solvent-exposed P4 residue was found to be important. Transplantation of the HSV-8 P2 or P4 residues onto the OVA-8 backbone created variant peptides O2S (P2I-->S) and O4E (P4N-->E) that bound considerably better to H-2Kbm8 than OVA-8. Furthermore, the double-substituted peptide, O2S4E, bound even better, revealing a cooperative effect of the two residues. The reciprocally substituted peptides H2I and H4N, generated by grafting the OVA-8 P2 and P4 residues onto the HSV-8 backbone respectively, bound to H-2Kbm8 slightly worse than HSV-8 but the double-substituted peptide H2I4N bound as poorly as OVA-8. Effects exerted by the P4 residue, which is solvent accessible and therefore available for the TCR contact, demonstrated that exposed peptide residues can, in certain situations, influence not only the TCR contact but also MHC-peptide binding.