The Src-homology 2 domain-containing, leukocyte-specific phosphoprotein of 76 kDa (SLP-76) is a hematopoietic adaptor that plays a central role during immunoreceptor-mediated activation of T lymphocytes and mast cells and collagen receptor-induced activation of platelets. Despite similar levels of expression in macrophages, SLP-76 is not required for Fc receptor for immunoglobulin G (IgG; FcgammaR)-mediated activation. We hypothesized that the related adaptor SLP-65, which is also expressed in macrophages, may compensate for the loss of SLP-76 during FcgammaR-mediated signaling and functional events. To address this hypothesis, we examined bone marrow-derived macrophages (BMM) from wild-type (WT) mice or mice lacking both of these adaptors. Contrary to our expectations, SLP-76(-/-) SLP-65(-/-) BMM demonstrated normal FcgammaR-mediated activation, including internalization of Ig-coated sheep red blood cells and production of reactive oxygen intermediates. FcgammaR-induced biochemical events were normal in SLP-76(-/-) SLP-65(-/-) BMM, including phosphorylation of phospholipase C and the extracellular signaling-regulated kinases 1 and 2. To determine whether macrophages functioned normally in vivo, we infected WT and SLP-76(-/-) SLP-65(-/-) mice with sublethal doses of Listeria monocytogenes (LM), a bacterium against which the initial host defense is provided by activated macrophages. WT and SLP-76(-/-) SLP-65(-/-) mice survived acute, low-dose infection and showed no difference in the number of liver or spleen LM colony-forming units, a measure of the total body burden of this organism. Taken together, these data suggest that neither SLP-76 nor SLP-65 is required during FcgammaR-dependent signaling and functional events in macrophages.