ABSTRACT We have previously shown that the addition of exogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) to nonactivated mouse peritoneal macrophages (MPM) limits Trypanosoma cruzi infections in vitro (E. Olivares Fontt and B. Vray, Parasite Immunol. 17:135–141, 1995). Lower levels of infection were correlated with a higher level of production of tumor necrosis factor alpha (TNF-α) in the absence of nitric oxide (NO) release. These data suggested that GM-CSF and/or TNF-α might have a direct parasitocidal effect on T. cruzi trypomastigotes, independently of NO release. To address this question, T. cruzi trypomastigotes were treated with recombinant murine GM-CSF (rmGM-CSF), recombinant murine TNF-α (rmTNF-α), or both cytokines in a cell-free system. Treatment with rmGM-CSF but not rmTNF-α caused morphological changes in the parasites, and most became spherical after 7 h of incubation. Both cytokines exerted a cytolytic activity on the trypomastigotes, yet the trypanolytic activity of rmTNF-α was more effective than that of rmGM-CSF. Viable rmGM-CSF- and rmTNF-α-treated parasites were less able to infect MPM than untreated parasites, and this reduction in infectivity was greatest for rmGM-CSF. Treatments with both cytokines resulted in more lysis and almost complete inhibition of infection. The direct parasitocidal activity of rmTNF-α was inhibited by carbohydrates and monoclonal antibodies specific for the lectin-like domain of TNF-α. Collectively, these results suggest that cytokines such as GM-CSF and TNF-α may directly control the level of T. cruzi trypomastigotes at least in vitro and so could determine the outcome of infection in vivo.