We analyzed the biological role of nitric oxide (NO) during murine Trypanosoma cruzi infection. Infection of mice with T. cruzi markedly increased NO synthesis. Administration of N-nitro-L-arginine methyl-esther (L-NAME) intraperitoneally or intragastrically diminished endogenous NO synthesis and resistance of mice to acute infection with three biologically different strains of T. cruzi. Mice protected against challenge with T. cruzi by transfer of T-cell-enriched populations from chronically infected animals, showed higher serum nitrate levels than controls non-transferred, or transferred, with T cells from non-immune mice. Administration of L-NAME abrogated transfer of resistance, suggesting NO participation in this process. Depletion of T cells from the transferred population abolished both protection and NO3- increase. On the contrary, mice chronically infected with T. cruzi showed no increased parasitemia or death upon treatment with L-NAME. The NO donor drug S-nitroso-acetyl-penicillamine was able to kill tissue culture or bloodstream trypomastigotes in vitro at biologically relevant concentrations. Conversely, NO appeared not to play a role in formation of inflammatory foci during T. cruzi infection, since infected mice treated with L-NAME showed no reduced inflammation.