Ly49 receptors in rodents, like KIR in humans, play an integral role in the regulation of NK cell activity. Some inhibitory Ly49 are known to interact with specific MHC I alleles to maintain tolerance to self tissues, and NK activation is triggered upon the loss of inhibitory signals due to pathological downregulation of self MHC I. Although a virally encoded ligand has been identified that can trigger NK cytotoxicity through an activating Ly49, some activating Ly49 also recognize MHC I and the role of most activating receptors in NK effector function remains poorly defined. As many Ly49 remain orphan receptors, we describe methods to unambiguously discern receptor–ligand pairs. Additionally, we describe a method for the mutagenesis of Ly49 and MHC ligands that can be used to define the motifs conferring receptor specificity for their ligands. Further elucidation of Ly49 ligands is required to continue to define the role of Ly49 in regulating NK cell effector function and may give vital clues to the role of KIR in human health and disease.