Following CNS trauma or ischemia, peroxynitrite may be a toxic intermediate which forms in vivo when nitric oxide condenses with superoxide. Alone, peroxynitrite appears to directly react with aromatic and sulfhydryl nucleophiles. However, at physiological pH, peroxynitrite is protonated and, in that form, will rapidly (within seconds) decompose to species with hydroxyl radical and nitrogen dioxide characteristics. These reactive species are shown to initiate lipid peroxidation, hydroxylate aromatic residues, and nitrate aromatic residues. This reactivity may contribute to differential toxicity in vivo and in vitro. Tirilazad mesylate (TZ) is a lipid-soluble antioxidant shown to inhibit iron-dependent lipid peroxidation. It is an effective therapy in a variety of CNS injury and ischemia models and is currently undergoing human clinical evaluation in stroke, head injury, and spinal injury. This study was designed to investigate the cytoprotective properties of TZ in a cerebellar granule cell model of peroxynitrite toxicity. Cytoprotective efficacy of TZ was based on viability measurements, blockade of lipid hydroperoxide generation, and blockade of nitrotyrosine formation. Cell viability was determined by [3H]-aminoisobutyric acid (3H-AIB) uptake, and lipid hydroperoxide and nitrotyrosine content were determined by HPLC assays. Tirilazad mesylate was found to have similar cytoprotective effects (approximately 50% protection at 100 microM) when applied before or after exposure of cells to peroxynitrite. In contrast, post-treatment with superoxide dismutase (50 units/ml) or allopurinol (100 microM) failed to produce any cytoprotection. Furthermore, we discovered that TZ inhibited the peroxynitrite-induced increase of phosphatidylethanolamine hydroperoxide (PEOOH), but did not affect the peroxynitrite-induced formation of nitrotyrosine formation. This suggests that the ability of TZ to afford cytoprotection in this peroxynitrite toxicity model is due to the inhibition of membrane-localized lipid peroxidation, and not to the inhibition of nitration of tyrosine residues.