MHC class I molecules can prevent NK cell-mediated cytotoxicity by interacting with inhibitory receptors on the effector cells. Different conclusions have been reached regarding possible peptide selectivity of these receptors. To address whether peptide selectivity is an exclusive feature of human or immunoglobulin-superfamily receptors, we have studied a system based on the murine NK receptor Ly-49C in the lectin-superfamily. Loading of TAP-deficient RMA-S cells with the H-2Kb-restricted, ovalbumin-derived peptide OVA(257 - 264) (pOVA) induced their ability to bind Ly-49C-transfected reporter cells, and also protected them from killing by Ly-49C+ NK cells. Other peptides that bound and stabilized H-2Kb equally well differed in their NK protective capacity. Comparison of the MHC class I peptide complexes (crystal structures and molecular models) revealed a conformational motif encompassing the C-terminal parts of the alpha1 helix (73 - 77) and the bound peptide that was common for the protective complexes. Substitution analysis of pOVA suggested that position 7 in the peptide may be critical for optimal protection as well as for the conformational motif at position 73 - 77. In conclusion, protection mediated by the murine C-type lectin receptor Ly-49C is peptide dependent and selective.