CD69, a member of the natural killer cell gene complex family of signal transducing receptors, represents one of the earliest activation antigens in human and murine lymphocytes. In contrast, human monocytes may express CD69 in a constitutive fashion. We have evaluated the expression and function of CD69 in murine bone marrow-derived macrophages. CD69 expression as determined by flow cytometry was not constitutive but was induced by stimulation with interferon-gamma (IFN-gamma) plus bacterial lipopolysaccharide (LPS) or tumor necrosis factor a (TNF-alpha). Stimulation with LPS alone was equally effective. Infection with the protozoan parasite Leishmania did not induce CD69 expression nor influence CD69 up-regulation by IFN-gamma plus LPS. Induction of CD69 expression was significantly inhibited in the presence of prostaglandin E2 or dibutyryl-cAMP. Stimulation of macrophages with anti-CD69 monoclonal antibody in the presence of IFN-gamma induced both nitric oxide production and TNF-alpha release. Moreover, anti-CD69 stimulation of Leishmania-infected macrophages resulted in elimination of the intracellular parasite. These results suggest that CD69 is an activation antigen for murine macrophages and may serve as a signaling receptor for an as yet uncharacterized ligand.