We have generated a monoclonal antibody (STOK2) which reacts with an inhibitory MHC receptor on a subset of alloreactive NK cells in the rat. This receptor, termed the STOK2 antigen (Ag), belongs to the Ly-49 family of lectin-like molecules and displays specificity for the classical MHC class I molecule RT1-A1c of PVG rats. Here, we have investigated the influence of the MHC on the selection, phenotype and function of the STOK2+ NK subset in a panel of MHC congenic and intra-MHC recombinant strains. STOK2 receptor density was influenced by the presence of its classical MHC I ligand RT1-A1c, as evidenced by a reduction of STOK2 Ag on the surface of NK cells from RT1-A1c+, as compared with RT1-A1c-, strains. In addition, a role for nonclassical MHC I RT1-C/E/M alleles in the selection of the STOK2 Ag was demonstrated. The relative number of STOK2+ NK cells was fivefold higher in rats expressing the RT1-C/E/M(av1) as compared with those expressing the RT1-C/E/M(u) class Ib haplotype. The STOK2 ligand RT1-A1c inhibited cytotoxicity of STOK2+ NK cells regardless of effector cell MHC haplotype. Allospecificity of STOK2+ NK cells varied markedly with effector cell MHC, however, and suggested that inhibitory MHC I receptors apart from STOK2 were variably co-expressed by these cells. These data provide evidence for the MHC-dependent regulation of the allospecific repertoire within a subset of potentially autoreactive Ly-49+ rat NK cells.