We produced a novel hamster monoclonal antibody (MAb), 14B11, that recognizes the majority of mouse natural-killer (NK) cells. Transfection studies demonstrated that 14B11 MAb binds a subset of Ly49 receptors, including three putative inhibitory receptors, Ly49F, I, and C. No binding to Ly49A, B, D, or G was detected. In addition, 14B11 was shown to bind the putative activating receptor Ly49H, which required co-transfection of the signaling molecule DAP12 for detectable cell surface expression. Thus, 14B11 is the first reported MAb to bind Ly49H and F. At the functional level, 14B11 MAb enhanced the lysis by IL-2 activated NK cells of an FcR+ target cell line (Daudi), but not an FcR- target cell (EL-4). Because F(ab')2 fragments of 14B11 failed to enhance lytic activity, the enhancement of lysis by intact antibody is apparently due to "redirected lysis," in which stimulatory receptors on the NK cell are bridged by antibody to Fc receptors on the target cell. Cell separation experiments demonstrated that the 14B11-dependent redirected lysis was markedly increased using NK cell populations that had been depleted of Ly49F,+ I,+ or C+ NK cells. Because such depletions are expected to enrich for Ly49H+ NK cells, these results suggest that the enhancement of lysis mediated by 14B11 MAb may be due to stimulation of the activating Ly49H receptor. In conjunction with other anti-Ly49 MAbs, the 14B11 MAb will be useful in further studies of Ly49 receptor function and specificity.