Macrophage cytotoxic response includes nitric oxide synthase- and NADPH oxidase-dependent production of nitric oxide (●NO) and superoxide (O2●-). Both enzymes use O2 as a substrate; therefore, their activity, as well as the formation of the product of the reaction between ●NO and O2●-, peroxynitrite (ONOO-), could be affected by local concentrations of O2. Moreover, O2 partial pressure (pO2) could modulate ●NO formation as it is demonstrated that iNOS expression is regulated by the hypoxia inducible factor 1-alpha (HIF-1α). While tissular pO2 varies between 2-15 %, most in vitro experiments are usually performed at a pO2 of 21%. To determine the effects of O2 concentration, J774.A1 macrophages were exposed during 1h or 24h to different O2 concentrations, ranging from 2 to 21%, and O2-●, ●NO and ONOO- production rates were measured. Also, to evaluate the influence of O2 as a signaling agent, HIF-1α activation and iNOS expression were assessed under the same conditions. Our results show that O2-● and ●NO production decreases when pO2 is under 10%, and therefore, less ONOO- production is detected. For instance, at 6% O2, almost 100% of O2-● formation is preserved, but the production of ●NO and ONOO- decreased to ≈60% of that observed in atmospheric air. After a 1h-incubation, no changes were observed in HIF-1α activation or iNOS expression. To evaluate the cytotoxic response, macrophage infection by Trypanasoma Cruzi was used as a model at different O2 concentrations. In accordance with our data, macrophages are less capable of eliminating parasites at a pO2 of 6% when compared to those incubated at 21%. However, even at 6% O2, macrophage activation to produce ONOO- increases 30% its capacity to eliminate T. Cruzi. Altogether, our results show the relevance of ONOO- as a cytotoxic agent at physiological pO2 conditions.