Peroxynitrite is formed by the reaction of nitric oxide (NO) and superoxide. Since widespread peroxynitrite activity was observed during experimental allergic encephalomyelitis (EAE), the effect of this strong lipid-peroxidizing agent on myelin integrity was examined. Incubation of myelin suspensions with the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) resulted in the formation of the lipid peroxidation product, malondialdehyde (MDA). MDA formation was inhibited in the presence of butylated hydroxytoluene, which interrupts the progression of the lipid peroxidation chain reaction. Superoxide dismutase inhibited the effect of SIN-1, which indicates a role for superoxide, and contradicts a role for its dismutation product, hydrogen peroxide. The latter was confirmed by the failure of the catalase to inhibit MDA formation. Neither NO nor superoxide alone induced significant MDA formation in myelin, indicating that peroxynitrite formation is required for myelin-lipid peroxidation. Interestingly, NO actually inhibited lipid peroxidation in myelin, as demonstrated using simple NO donors. On the other hand, the simultaneous production of superoxide, as achieved with the NO-donor SIN-1, negated the inhibitory effect of NO. Finally, the production of isoprostanes, novel products generated during lipid peroxidation, was examined. Peroxynitrite-induced peroxidation of myelin resulted in isoprostane formation. Furthermore, increased levels of F2-isoprostanes and neuroprostanes were observed in spinal cords of mice during early progressive stages of autoimmune encephalomyelitis.