Natural killer (NK) cells are important effectors of innate immunity. In contrast to many studies of interleukin-2 (IL-2)-activated NK cells, the physiologic requirements for stimulating resting NK cells have only recently received attention. Given the emerging variety of dendritic cell (DC) types and their division of labor for stimulating immunity, we compared the capacity of monocyte-derived DCs (moDCs) with that of CD34⁺ hematopoietic progenitor cell (HPC)-derived dermal-interstitial DCs (DDC-IDCs) and Langerhans cells (LCs) to stimulate resting NK cells. MoDCs, and to a lesser extent CD34⁺ HPC-derived DDC-IDCs, directly stimulate NK-cell proliferation, CD56 up-regulation, and cytotoxicity. LCs, on the contrary, require exogenous IL-2 or IL-12 to activate NK cells, but they can maintain resting NK-cell viability and sustain NK-cell proliferation induced by moDCs. LCs do not secrete bioactive IL-12p70 but do produce significantly higher concentrations of IL-15 and IL-18 than either of the other 2 DC types. Despite secretion of IL-15, LCs lack IL-15R-α for surface presentation of IL-15. This together with the deficiency of IL-12p70 undermines any direct NK-cell activation by LCs. Hence, the principal myeloid DCs differ in critical ways regarding the stimulation of NK and T lymphocytes and could be used or targeted accordingly in DC-based immunotherapies. (Blood. 2005;105:266-273)