The MHC class I molecule H-2Dd (Dd) acts as a ligand for the inhibitory NK cell receptor Ly-49A. We have constructed altered Dd molecules by site-directed mutagenesis, replacing residues with the corresponding amino acids from the Db molecule, which fails to inhibit via Ly-49A. Mutations at positions 73 and 156 (DdS73WD156Y) impaired the protective effect of the Dd molecule, as evaluated by testing lymphoma cells transfected with the mutant gene for sensitivity to killing by Ly-49A+ NK cells in vitro and rejection by NK cells in vivo. The altered residues form a hydrophobic ridge across the floor of the antigen binding cleft. A mutation in the alpha helix of the alpha2 domain, facing the solvent and without direct contact with the peptide (DdA150S) had no effect. Dd recognition by Ly-49A+ NK cells is considered to be peptide dependent, but not peptide specific. Our results indicate that alterations of residues buried in the antigen binding cleft can induce changes in peptide binding patterns and/or conformational changes in the Dd molecule that make the trimolecular complex less permissive for inhibition of Ly-49A+ NK cells.