Donor NK cells have been shown to be able to promote engraftment during allogeneic bone marrow transplantation. They could specifically suppress or delete host reactive cells, thereby facilitating engraftment of donor marrow. To further elucidate the mechanism, we showed that activated H2(d) ALAK cells (adherent lymphokine activated killer, IL-2 activated T cell-depleted bone marrow and spleen cells) from BALB/c mice significantly suppressed the proliferation of H2(b) splenocytes from C57BL/6 mice in mixed lymphocyte responses (MLR) stimulated with irradiated H2(d) splenocytes from BALB/c mice (P < .01). The ability for H2(b) splenocytes to kill H2(d) tumor targets was also significantly inhibited by activated H2(d) ALAK cells (P < .01). The same number of H2(b) ALAK cells or H2(d) splenocytes did not show the same suppressive effect. These results suggested that activated H2(d) ALAK cells could specifically suppress the anti-H2(d) activity of the H2(b) splenocytes. Anti-tumor growth factor (TGF)beta antibody blockade did not diminish this suppressive effect of ALAK cells, suggesting that this activity is not dependent on TGF-beta secretion. ALAKs from gld (FasL mutant) mice suppressed the allo-responses as well as the wild-type ALAK cells. The ALAKs from pfp (perforin knockout) mice did not completely block the inhibitory effect, which suggested that the suppressive effect of the allogeneic ALAK cells could be partially caused by perforin-mediated killing. We further demonstrated that donor ALAK cells could promote engraftment by suppressing host alloreactive responses in a nonmyeloablative allogeneic BMT model. These studies suggest that activated donor NK cells specifically suppress the alloreactive cells and provide a promising way to promote donor engraftment without involving systemic and nonspecific suppression of the immune system.