Activation-induced cell death (AICD) of CD4+ T lymphocytes was described in infection with Trypanosoma cruzi, but a role for AICD in modulating parasite spread in host cells has not been investigated. In this study, replication of T. cruzi in vitro in murine macrophage (Mφ) monolayers was investigated. Long term (5 to 13 day) replication of infective (trypomastigote) T. cruzi forms was blocked by supernatants from activated (anti-TCR) CD4+ T cells of infected mice or by rIFN-γ. However, when CD4+ T cells from infected mice were cocultured with Mφ and activated by anti-TCR, marked exacerbation of trypomastigote growth in Mφ ensued. The deleterious effect required contact between T cells and infected Mφ. Both anti-Fas and TCR activation killed a proportion of CD4+ T cells. Ly-6 activation did not induce AICD and did not exacerbate parasite growth. However, Fas-mediated killing of T cells before Ly-6 activation led to exacerbated parasite growth. Although a minor population, Fas-susceptible cells were the major source of IFN-γ production by activated T cells. Addition of a neutralizing anti-Fas ligand antibody blocked 50 to 60% of CD4+ T cell AICD and reduced trypomastigote growth in T/Mφ cocultures stimulated by anti-TCR. The results demonstrate that in CD4+ T cells from infected mice, the onset of AICD selectively ablates IFN-γ production and up-regulates parasite replication in Mφ in vitro. These findings suggest a deleterious role for AICD in T. cruzi infection.