ABSTRACTNatural killer (NK) cells are important components of a protective immune response against intracellular pathogens such asLeishmaniaparasites, which reside within myeloid cells. Previousin vivostudies in murine cutaneous or visceral leishmaniasis showed that NK cells are activated by conventional dendritic cells in a Toll-like receptor 9-, interleukin-12 (IL-12)-, and IL-18-dependent manner during the early phase of infection and help to restrict the tissue parasite burden by unknown mechanisms. Here, we tested whether NK cells contribute to the control ofLeishmaniainfections by lysing or by activating infected host cells. Coculture experiments revealed that activated NK cells from poly(I:C)-treated mice readily killed tumor target cells, whereasLeishmania infantum- orL. major-infected macrophages or dendritic cells remained viable. Infection withLeishmaniadid not significantly alter the expression of NK cell-activating molecules (retinoic acid early transcript alpha [Rae-1α], mouse UL16-binding protein-like transcript 1 [MULT-1], CD48) or inhibitory molecules (major histocompatibility complex [MHC] class I, nonclassical MHC class 1b molecule Qa-1) on the surface of myeloid cells, which offers an explanation for their protection from NK cell cytotoxicity. Consistent with thesein vitrodata,in vivocytotoxicity assays revealed poor cytolytic activity of NK cells against adoptively transferred infected wild-type macrophages, whereas MHC class I-deficient macrophages were efficiently eliminated. NK cells activated by IL-12 and IL-18 stimulated macrophages to kill intracellularLeishmaniain a cell contact-independent but gamma interferon-, tumor necrosis factor-, and inducible nitric oxide synthase-dependent manner. We conclude thatLeishmaniaparasites, unlike viruses, do not render infected myeloid cells susceptible to the cytotoxicity of NK cells. Instead, soluble products of NK cells trigger the leishmanicidal activity of macrophages.