Macrophages produce and release superoxide anion (O2.-) and nitric oxide (.NO) as part of their microbicidal effector molecules. The simultaneous production of O2.- and .NO results in the rapid formation of peroxynitrite anion (ONOO-) by macrophages. Peroxynitrite is a strong oxidant with a half-life of less than 1 s in biological systems. There is solid experimental evidence implicating .NO and O2.- in macrophage-induced cytotoxicity against bacteria, parasites, and tumor cells. However, the cytotoxic role of peroxynitrite in these processes remains to be studied. In this work we demonstrate the parasiticidal activity of ONOO- against Trypanosoma cruzi. Peroxynitrite was highly trypanocidal, killing T. cruzi in a dose-dependent manner. Addition of 500 microM ONOO- as a single bolus resulted in 50% inhibition of cell proliferation as followed by growth curves. Fifty percent inhibition of [3H]thymidine incorporation measured at 6 h postaddition of ONOO- was obtained at 150 microM. Addition of ONOO- as a continuous infusion rather than a single bolus resulted in an even stronger inhibition in cell growth. Other cytotoxic effects of ONOO- included cellular swelling and inhibition of cell motility. Classical hydroxyl radical scavengers and metal chelators afforded minimal protection against ONOO(-)-mediated cytotoxicity, indicating that peroxynitrite anion itself, rather than the .OH-like oxidant derived from its proton-catalyzed decomposition, was the main damaging species. From literature data we estimated the production of ONOO- by activated macrophages inside phagolysosomes to be around 500 microM/min. Therefore, our results demonstrate that ONOO- may operate in vivo as a critical macrophage-derived reactive intermediate against T. cruzi.