Mouse NK cells express Ly-49 receptors specific for classical MHC class I molecules. Several of the Ly-49 receptors have been characterized in terms of function and ligand specificity. However, the only Ly-49 receptor-ligand interaction previously described in detail is that between Ly-49A and H-2D(d), as studied by point mutations in the ligand and the crystal structure of the co-complex of these molecules. It is not known whether other Ly-49 receptors bind MHC class I in a similar manner as Ly-49A. Here we have studied the effect of mutations in Ly-49C on binding to the MHC class I molecules H-2K(b), H-2D(b), and H-2D(d). The MHC class I molecules were used as soluble tetramers to stain transiently transfected 293T cells expressing the mutated Ly-49C receptors. Three of nine mutations in Ly-49C led to loss of MHC class I binding. The three Ly-49C mutations that affected MHC binding correspond to Ly-49A residues that are in contact or close to H-2D(d) in the co-crystal, demonstrating that MHC class I binding by Ly-49C is dependent on residues in the same area as that used by Ly-49A for ligand contacts.