We address the mechanism of hybrid resistance (HR) in vitro using NK effector cells and target lymphoblasts from H-2b, H-2d, and H-2b/d mice. The 5E6 (Ly49C)+ subset of F1 NK cells lyse BALB/c (H-2d) but not B6 (H-2b) targets unless either anti-5E6 or anti-H-2Kb MAbs are present. H-2Dd transgenic B6 (D8) targets are not susceptible to F1 Ly49A+ effectors. Furthermore, 5E6+ Ly49A+ F1 effectors lyse B6 and BALB/c targets only in the presence of anti-5E6 and anti-Ly49A MAbs, respectively. Thus, recognition of H-2Kb by 5E6 and H-2Dd by Ly49A transduce independent inhibitory signals. Moreover, anti-5E6 MAbs enable 5E6+ BALB/c NK cells to lyse (BALB/c x B6)F1 targets. These data support the "missing self" and not the "hemopoietic histocompatibility antigen" hypothesis for HR. In addition, 5E6+ NK cells from BALB/c and BALB.B, but not B6 or (BALB/c x B6)F1, mice receive negative signals from both H-2d and Kb class I antigens. Thus, allelic differences in 5E6 (C57BL versus BALB) may regulate recognition events by NK cells.