The generation and activity of NK cells appear to be regulated by a particular set of cytokines. We examined the in vivo effects of recombinant human Flt3 ligand (Flt3-L), a recently cloned potent hemopoietic cytokine, on NK cell development in mice. Daily ip administration of Flt3-L consistently induced striking increases in both the absolute number and the total cytotoxic activity of mature nonactivated NK cells within various tissues. Dose-and time-dependent increases were observed in the bone marrow (∼ 2-and∼ 11-fold, respectively), thymus (∼ 2.8-and∼ 2.0-fold), blood (∼ 11-and∼ 15-fold), spleen (∼ 10-and∼ 9-fold), and liver (∼ 15-and∼ 39-fold). In addition, IL-2 induced a rapid increase in NK activity, NK cell proliferative responses, generation of lymphokine-activated killer activity, and development of activated adherent NK cells, which were all significantly increased by Flt3-L treatment. Thus, in addition to its recently reported capacity to stimulate dendritic cell production, Flt3-L has a prominent biologic role in NK cell generation in vivo. This is probably a result of selectively induced expansion of NK cell progenitors (pro-NK cells), because Flt3-L stimulates in vitro proliferation of pro-NK cells without affecting the cytotoxicity of mature NK cells. The results also indicate that either alone or in combination with a potent activator of NK cells, such as IL-2, Flt3-L could be used to markedly augment the number and activity of NK cells, especially in the liver. Flt3-L appears to have considerable potential for therapy of both cancer and viral infection.