The ability of natural killer (NK) cells to eliminate normal allogeneic hemic cells is well established in several species including mice, rats, and humans. The controlling elements for NK susceptibility in these species map to the major histocompatibility complex (MHC), but in contrast to findings in mice and humans, the mode of inheritance is not always recessive in rats. This finding is not easily explained by the missing self and hemopoietic histocompatibility (Hh) models for NK recognition, and has led to the idea that certain alloantigens may trigger NK cell reactivity. In our in vitro system for assessing rat NK alloreactivity, we have employed target and inhibitor cells from a large panel of MHC congenic, intra-MHC recombinant and MHC mutant rat strains, as well as appropriate F1 hybrids between them, and we show the following: (a) The nonclassical class I (RT1.C) region was most important in determining the susceptibility of target cells to alloreactive NK cells in vitro. Lymphocyte susceptibility to lysis in vivo also mapped to the C region, which supports the concept that the in vivo and in vitro alloreactivity assays reflect the same recognition process. (b) Four different RT1-controlled NK allospecificities (represented by the u, l, a, and n haplotypes) could be discerned when we used polyclonal NK cells from the PVG (RT1c) strain as effector cells. Three of the target specificities recognized were controlled mainly by the RT1.C region. (c) The expression of RT1.C region-controlled parental strain NK allodeterminants could be demonstrated in F1 hybrids heterozygous for the C region alone and were therefore inherited nonrecessively. (d) Loss of an RT1.C region-controlled NK allospecificity could be shown with the MHC mutant LEW.1LM1 rat strain characterized by a genomic deletion of about 100 kb of the C region. Taken together, these observations have demonstrated a major importance of the nonclassical class I region, i.e., RT1.C, in controlling rat NK allorecognition, and have thereby assigned a hitherto undescribed immunological property to this region. Furthermore, some of the present data are consistent with the existence of polymorphic NK-triggering alloantigens that are coded for by the RT1.C region.