Sublethal injurious stimuli induce tolerance to subsequent lethal insults, a phenomenon termed preconditioning. Inducible nitric oxide synthase (iNOS) is essential for the preconditioning induced by transient bilateral common carotid artery occlusion (BCCAO) or by systemic administration of the endotoxin lipopolysaccharide (LPS). We used a model of brain injury produced by neocortical injection of N-methyl-d-aspartate (NMDA) to investigate the mechanisms by which iNOS-derived nitric oxide (NO) contributes to tolerance induced by LPS or BCCAO. We found that the tolerance is blocked by the iNOS inhibitor aminoguanidine, is not observed in iNOS-null mice, and is rescued by the NO donor DTPA NONOate. Lipopolysaccharide failed to induce preconditioning in mice lacking the nox2 subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, suggesting that superoxide derived from NADPH oxidase is needed for the induction of the tolerance. Because superoxide reacts with NO to form peroxynitrite, we investigated the role of peroxynitrite. We found that LPS induces the peroxynitrite marker 3-nitrotyrosine in cortical neurons and that the peroxynitrite decomposition catalyst FeTPPS abolishes LPS-induced preconditioning. These results suggest that the protective effect of iNOS-derived NO is mediated by peroxynitrite formed by the reaction of NO with NADPH oxidase-derived superoxide. Thus, peroxynitrite, in addition to its well-established deleterious role in ischemic brain injury and neurodegeneration, can also be beneficial by inducing tolerance to excitotoxicity.