Serum from mice infected with Trypanosoma cruzi-suppressed (=SSS) lipopolysaccharide (LPS)-, phytohemagglutinin (PHA)-, and concanavalin A (Con A)-induced lymphoblast transformation when added to cultures of spleen cells or lymph node cells. This serum maximally suppressed blastogenic responses in spleen cell and lymph node cell cultures that contained supportive fetal bovine serum concentrations of 2% and 4%, respectively. Preincubation of lymphoid cells with SSS for 18 to 48 hr prior to initiation of the blastogenesis assay led to suppression of LPS-, PHA-, and Con A-induced proliferation at the optimal concentration of supportive fetal bovine serum (5%), whereas adsorption of lymphoid cells with SSS at 4 C for 30 min before stimulation with mitogen led to suppression of LPS-induced proliferation in spleen cells only. There was a close temporal correspondence between the induction and manifestation of suppression to the T-cell mitogens (PHA and Con A), but the manifestation of suppression preceded the induction of suppression to the B-cell mitogen (LPS) by approximately 12 hr. The SSS-induced suppression of proliferative responses, except in spleen cell cultures stimulated with LPS, was shown to be dependent on the presence of macrophages during the preincubation and stimulation phases of the assay system. The combined results of experiments in which macrophages were preincubated with SSS, or in which macrophages from the spleen were cultured with lymphocytes from the lymph nodes and vice versa (before and after preincubation with SSS), clearly demonstrated the presence of SSS-activated suppressor cells in the spleen, but not in the lymph nodes. Furthermore, the activation of these suppressor macrophages was reliant upon interactions with splenic lymphocytes.