In multiple sclerosis (MS) the mechanisms of injury caused by peroxynitrite remain uncertain. To study histological, ultra structural and molecular alterations caused by peroxynitrite in brain, the peroxynitrite donor 3-morpholinosydnonimine was injected in rat corpus callosum. Peroxynitrite induces strong primary axonal damage with characteristics of primary acute axonopathy, together with severe myelin alteration, myelin vacuolation and demyelination, and nitrotyrosine formation as confirmed by detection of nitrosated target proteins. Administration of the peroxynitrite scavenger uric acid inhibited these effects. In vivo, peroxynitrite leads to a disorganisation of myelin and to axonal damage presenting some similarities to the formation of MS lesions. Understanding the action of peroxynitrite in this process will open new therapeutic strategies by specific inhibition of peroxynitrite formation and action.