Trypanosoma cruzi infection is associated with a severe unresponsiveness of spleen cells (SC) to antigens and mitogens. A high production of NO by concanavalin A (Con A)‐stimulated SC from infected but not from control mice was observed. Neutralization of endogenous IFN‐γ production or treatment with NO synthase (NOS) inhibitor, l‐N‐monomethyl‐arginine, blocked Con A‐induced NO production and greatly restored proliferation by SC from infected mice. This was confirmed by using IFN‐γR^–/– and inducible NOS (iNOS)^–/– knockout mice, since unresponsiveness to mitogens of SC from those infected mice was much less pronounced than in control littermates. Interestingly, SC unresponsiveness was associated with a huge increase in CD11b⁺ cells that express Ly‐6G (Gr1)⁺ and other immature myeloid markers These cells were absent in infected IFN‐γR^–/– spleens. Purified immature Gr1⁺CD11b⁺ cells produced NO and expressed iNOS upon IFN‐γ treatment, and were able to inhibit T cell proliferation. In addition, depletion of myeloid CD11b⁺ cells abrogated NO production and restored mitogen‐induced proliferation, but not IL‐2 synthesis, in SC from infected mice. IL‐2 production and CD25 cell surface expression by mitogen‐activated T cells were greatly depressed in SC from IFN‐γR^–/– and iNOS^–/– mice, confirming that Gr1⁺CD11b⁺ cells were not involved in their down‐regulation. In contrast, IL‐5, tumor necrosis factor and IFN‐γ production, and CD69 expression by T cells were not depressed in infected SC. The results indicate the existence of an immunosuppressive mechanism during T. cruzi infection, mediated through IFN‐γ‐dependent NO secretion by immature Ly‐6G (Gr1)⁺CD11b⁺ myeloid cells.