Abstract Natural killer (NK) cells kill tumors and infected cells by integrating signals from two functional types of receptors, activation and inhibitory. In the mouse, many of these receptors belong to the Ly49 family, encoded by a cluster of highly related genes, discovered by the applicant’s laboratory. The highly polymorphic Ly49 receptors are related to killer immunoglobulin-like receptors (KIRs) on human NK cells as outstanding examples of convergent evolution. The applicant’s laboratory previously showed that the Ly49H activation receptor is responsible for genetic resistance of C57BL/6 mice to murine cytomegalovirus (MCMV) infections. Ly49H recognizes a molecule encoded by MCMV, in a manner independent of major histocompatibility complex class I (MHC-I) alleles. Studies from other groups suggest that in non-C57BL/6 mice, other Ly49 activation receptors that are not alleles of Ly49H appear to recognize MCMV-infected cells in vitro but their in vivo significance has not been established. Ironically, a recent publication from the applicant’s lab also show that Ly49 inhibitory receptors can provide NK cell-mediated protection against MCMV. This process depends on MHC-I alleles, via Ly49 inhibitory receptor-dependent licensing or education of NK cells, and their capacity to detect loss of MHC-I expression, termed missing-self, during MCMV infection. These studies provide the basis for understanding the association of human inhibitory KIRs and their MHC-I ligands that are also paradoxically associated with protection from viral infection, indicating that further studies may have clinical significance. But it has been challenging to study these receptors further in vivo due to the complexities of the Ly49 receptors, including their polymorphism and variegated expression. Here the applicant presents preliminary data in which expression of all Ly49s have been extinguished and can be replaced by a single Ly49 expressed on all NK cells. This technological advance will facilitate the further elucidation of how NK cells control MCMV infection. Thus, the Specific Aims of this proposal are to: 1) Study non-Ly49H activation receptors in NK cell responses to MCMV. 2) Characterize NK cells with inhibitory receptors mediating MCMV control. 3) Evaluate interactions between Ly49 receptors in control of MCMV. Thus, these studies will provide new insight into the mechanisms by which NK cells control viral infection.