To explore the modulatory effects of IL-2-activated NK cells on hematopoietic stem cell (HSC) engraftment further, we used fresh newborn liver cells (NLC) and IL-2-activated newborn liver cells (ANLC) as combined sources, respectively, of transplanted HSC and IL-2-activated NK cells free of contaminating CD3+ T cells. As previously found with adult IL-2-activated spleen cells, NLC cultured with IL-2 for 7 days exhibited lymphokine-activated killer (LAK) activity, veto activity, and natural suppressor activity, and enhanced both short-term and long-term stem cell engraftment by intact co-injected syngeneic and allogeneic NLC in totally MHC-mismatched lethally irradiated recipients. However, unlike adult IL-2-stimulated adult spleen cells, IL-2-activated NLC lacked CD3+ T cells and failed to induce lethal GVHD. FACS analysis and cell sorting experiments showed that the cells in ANLC which enhanced short-term HSC engraftment belonged to the relatively immature CD3-NK1.1-2B4+ NK cell subset. By contrast, cells belonging to the more mature CD3-NK1.1+2B4+ NK cell subset showed no HSC-enhancing effects. Identification and isolation in humans of similar NK cell enhancers of HSC could lead to a new approach to improving stem cell engraftment in MHC-mismatched recipients without increasing the risk of GVHD.