The role of cytokines in the control of tissue parasitism and pathogenesis of experimental Chagas’ disease was investigated. Wild-type and different cytokine as well as inducible nitric oxide synthase (iNOS) knockout mice were infected with the Colombian strain of Trypanosoma cruzi, and the kinetics of tissue parasitism, inflammatory reaction, parasitemia, and mortality were determined. We demonstrate the pivotal role of the interleukin (IL)-12/interferon (IFN)- /iNOS axis and the antagonistic effect of IL-4 in controlling heart tissue parasitism, inflammation, and host resistance to acute infection with T. cruzi. Further, the heart and central nervous system were shown the main sites of reactivation of T. cruzi infection in mice lacking functional genes for IFN- and IL-12, respectively. Our results also show that in contrast to IFN- knockout (KO) mice, splenocytes from IL-12 KO mice infected with T. cruzi produced low levels of IFN- upon stimulation with antigen. Consistently, high levels of anti-T. cruzi IgG2a antibodies were detected in the sera from IL-12 KO, but not from IFN- KO mice, infected with the Colombian strain of T. cruzi. Thus, our results suggest that the level of IFN- deficiency is a major determinant of the site of reactivation of T. cruzi infection in immunocompromised host.