The main functions of natural killer (NK) cells are early protection against viruses or tumor cells and production of cytokines that regulate immune functions. The present study assessed the role of different NK subsets in exerting graft-versus-leukemia effects in recipients of human leukocyte antigen (HLA) haploidentical hematopoietic transplants and monitored for the first time CD3–/CD56–lymphocyte expansion. CD3–/CD56–cells expressed NK cell-associated molecules, such as CD16, NKp46, NKp30, CD244 (2B4), CD161, and killer cell immunoglobulin-like receptors. CD3–/CD56–cells further exhibited the classical functional characteristics of NK cells: cytolysis of target cells lacking HLA class I, antibody-dependent cellular cytotoxicity and cytokine production. These results demonstrate that CD56–NK cells are functional, recognize missing self and, like their CD56+ counterparts, may contribute to graft-versus-leukemia reactions.