IFN-γ has long been recognized as a cytokine with potent and varied effects in the immune response. Although its effects on specific cell types have been well studied in vitro, its in vivo effects are less clearly understood because of its diverse actions on many different cell types. Although control of multiple protozoan parasites is thought to depend critically on the direct action of IFN-γ on macrophages, this premise has never been directly proven in vivo. To more directly examine the effects of IFN-γ on cells of the macrophage lineage in vivo, we generated mice called the “macrophages insensitive to IFN-γ”(MIIG) mice, which express a dominant negative mutant IFN-γ receptor in CD68+ cells: monocytes, macrophages, dendritic cells, and mast cells. Macrophage lineage cells and mast cells from these mice are unable to respond to IFN-γ, whereas other cells are able to produce and respond to this cytokine normally. When challenged in vitro, macrophages from MIIG mice were unable produce NO or kill Trypanosoma cruzi or Leishmania major after priming with IFN-γ. Furthermore, MIIG mice demonstrated impaired parasite control and heightened mortality after T. cruzi, L. major, and Toxoplasma gondii infection, despite an appropriate IFN-γ response. In contrast, MIIG mice displayed normal control of lymphocytic choriomeningitis virus, despite persistent insensitivity of macrophages to IFN-γ. Thus, the MIIG mouse formally demonstrates for the first time in vivo, the specific importance of direct, IFN-γ mediated activation of macrophages for controlling infection with multiple protozoan parasites.