We investigated if an infusion of alloreactive natural killer (NK) cells would reduce GVHD and mediate antitumor effects in mice undergoing MHC-matched allogeneic stem cell transplantation (SCT). Balb/c mice bearing RENCA tumors underwent an allogeneic SCT from MHC-matched B10.d2 donors and were given a single infusion of either Ly49 ligand-matched, ligand-mismatched, or no donor NK cells. Recipients of Ly49 ligand-mismatched NK cells had a reduced incidence of graft-versus-host disease (GVHD; 39% vs 100%; P < .01), and prolonged survival (median 84 days vs 39 days; P < .01) compared with SCT recipients not receiving NK cells. Recipients of Ly49 ligand-matched NK cells had the same incidence of GVHD and similar survival compared with controls not receiving NK cells. Pulmonary tumor burden was significantly (P < .01) lower in recipients that received Ly49-mismatched or Ly49-matched NK cells compared with recipients not receiving NK cells. These data provide in vivo evidence that a single infusion of alloreactive donor NK cells reduces GVHD and mediates antitumor effects following MHC-matched allogeneic transplantation.