Murine natural killer (NK) cell subsets, as defined by expression of members of the Ly49 gene family, discriminate target cells expressing different major histocompatibility complex (MHC) class I alleles. For example, Ly49A+ NK cells lyse H-2b but not H-2d tumor target cells. The specificity arises because Dd on target cells binds to Ly49A, transducing an inhibitory signal into the Ly49A+ NK cells. The capacity of NK cells to discriminate allelic class I determinants raises a key issue: are NK cells self-tolerant, and if so what are the mechanisms that lead to self-tolerance? As previously reported, potentially autoaggressive Ly49A+ NK cells are not clonally deleted in H-2b mice. However, IL-2-cultured Ly49A+ effector cells from H-2b mice exhibit reduced lysis of H-2b (self) concanavalin A blast target cells, compared to Ly49A+ effector cells from H-2d mice. Possible mechanisms accounting for this self-tolerance are addressed in this report. Self-tolerance was not due to anergy of the cells, because the Ly49A+ effector cells from both types of mice lysed beta 2-microglobulin-deficient target cells efficiently and equivalently. These results also suggest that tolerance results from inhibition mediated by beta 2m-dependent H-2b class I molecules. Significantly, blockade of Ly49A on Ly49A+ effector cells from H-2b mice did not restore lysis of H-2b target cells, suggesting that inhibition is not mediated through the Ly49A receptor. Additional experiments suggest that inhibition is also not mediated primarily through the Ly49C receptor. These results suggest that Ly49A+ effector cells from H-2b mice, unlike those from H-2d mice, express inhibitory receptors specific for H-2b molecules that are distinct from Ly49A and Ly49C.