Peroxynitrite is formed by the reaction of superoxide with nitric oxide, an important neurotransmitter. Incubation of rat brain synaptosomes with peroxynitrite resulted in the consumption of antioxidant substances such as α-tocopherol, ascorbate, and thiols. Membrane cholesterol was not oxidized under the same conditions. α-Tocopherol and ascorbate in synaptosomes were oxidized very rapidly by peroxynitrite. In contrast, previous reports in the literature have shown that peroxynitrite treatment did not oxidize tocopherol in human plasma. Peroxynitrite in sufficient concentrations oxidized all of the tocopherol and ascorbate in synaptosomes. Thus, the oxidant is able to diffuse to the different membranes in synaptosomes and oxidize tocopherol in all of them. α-Tocopherol is converted quantitatively to tocopherolquinone during the oxidation. Significant amounts of thiols (at least 30% of the total thiols) do not seem to be accessible to oxidation by peroxynitrite. However, the concentration of thiols is much higher than those of tocopherol and ascorbate. Addition of the hydroxyl radical quenchers benzoate or mannitol or the enzymes superoxide, dismutase or catalase (alone or together) did not affect the oxidation of tocopherol and ascorbate by peroxynitrite, whereas cysteine and glutathione blocked the oxidation. Therefore, reactive oxygen species may not be directly involved as intermediates in oxidations induced by peroxynitrite. The latter is a potent oxidizing agent that can oxidize substances such as tocopherols, ascorbate, and thiols in the immediate vicinity of its formation. The antioxidant nutrients ascorbate and tocopherol could play important roles in protecting brain from oxidative damage induced by peroxynitrite.