An intense suppression of splenic T cell proliferation to mitogens and to Ags from the parasite is characteristic of the acute phase of Trypanosoma cruzi infection in mice. The impairment of proliferation is coincident with high levels of IFN-gamma and nitrite and decreased production of IL-2 in the supernatants of spleen cell cultures from infected mice. Previous work demonstrated that suppression of proliferation is largely mediated by the population of adherent cells in the infected spleen. In this study we confirmed the active suppression exerted by these cells on Con A, anti-CD3, and parasite Ag-stimulated proliferation of CD4+ splenic T cells. Inasmuch as the high production of IFN-gamma and of nitrite were compatible with intense macrophage activation and nitric oxide (NO) production, we determined the effects of cytokines that regulate macrophage activation and of NO on the proliferation of spleen cells from infected mice. We show that spleen cell proliferation to Ag and to T cell polyclonal stimuli is increased by neutralizing mAbs to IFN-gamma, TNF-alpha and -beta, or by the inhibitor of NO synthase, NG-monomethyl-L-arginine, added to the cultures. The addition of rIL-2 or rIL-4 also contributed to suppression reversal, and the combined addition of rIL-2 and anti-IFN-gamma mAb further increased lymphocyte proliferation. Anti-IL-4, anti-IL-10, or anti-TGF-beta neutralizing mAbs did not modify suppressed proliferative responses, and the addition of rIL-10 or of rTGF-beta also did not recover cell proliferation. Thus, the suppression of proliferative responses in T. cruzi-infected mice resulted largely from increased NO production by macrophages activated by IFN-gamma and TNF allied to insufficient IL-2 to fully support in vitro growth of T lymphocytes.