Mice infected with the protozoan parasite, Trypanosoma cruzi, are known to be immunosuppressed in responsiveness to heterologous antigens and parasite-specific antigens. This suppression is mediated by suppressor macrophages and is exemplified by deficient T cell activity and abnormal cytokine production. Neither the mechanism by which suppressor macrophages effect suppression nor the characteristics of these suppressor macrophages is known. In the present study, we analyzed the regulatory cell populations in splenocytes of infected mice (SCinf) and their interactions by limiting dilution-partition analysis, an approach which allows the functional separation of multiple regulatory cell subpopulations within cell mixtures. Our results demonstrate the presence of a complex immunoregulatory circuit in SCinf affecting the generation of anti-sheep erythrocyte antibody responses in vitro. Titration of SCinf (but not peritoneal exudate or lymph node cells) into Mishell-Dutton microcultures of normal spleen cells generated complex dose-response curves with two zones of suppressed responses following the addition of either low or high doses of SCinf to the cultures. Addition of intermediate doses of SCinf to the microcultures restored responsiveness. Both the low- and high-dose zones of suppression were shown to be mediated by macrophages, whereas T cells were responsible for the restored responsiveness at intermediate doses of SCinf. Examination of the development of this complex regulatory pattern during the course of the acute phase of infection indicated the sequential development of one suppressor macrophage population, followed by the development of the beneficial T cell population, and finally the expression of the second suppressive macrophage population.