The relationship between nitric oxide (N = O) produced by mouse peritoneal macrophages (MPM) and Trypanosoma cruzi infection is still poorly understood. The conditions of MPM activation by gamma‐interferon (IFN‐γ) to trigger a N = O‐dependent trypanocidal activity, as well as the effect of parasite infection or of reactive oxygen species (ROS) inhibitors on the N = O release were studied. T. cruzi infection occurring after a previous 24 h MPM activation induced an enhancement of nitrite levels (the stable degradation product of N = O) in cell supernatants; both the percentage of infected MPM and the number of amastigotes per infected cell were decreased in comparison to infected but non‐activated MPM. Addition of superoxide dismutase or catalase to non‐infected but activated MPM increased the nitrite levels; these were not detectable when L‐arginine inhibitors were added together with ROS inhibitors. The latter had no effect on infection nor on nitrite levels when infection occurred after pre‐activation, and induced only a weak nitrite release when infection took place before MPM activation. Altogether, these results support the involvement of N = O in the inhibition of T. cruzi infection by IFN‐γ‐preactivated macrophages, together with the upregulation of N = O release by T. cruzi infection independently of the respiratory burst.