The pathophysiology of brain lesions associated with cerebral palsy is multifactorial and likely involves excess release of glutamate and excess production of free radicals, among other factors. Theoretically, antioxidants could limit the severity of these brain lesions. Peroxiredoxins are a family of peroxidases widely distributed in eukaryotes and prokaryotes. Peroxiredoxin 5 (PRDX5) is a recently discovered mammalian member of this family of antioxidant enzymes that is able to reduce hydrogen peroxide and alkyl hydroperoxides. The present study was designed to examine the neuroprotective effects of recombinant PRDX5 against neonatal excitotoxic challenge in both in vivo and in vitro experiments. For in vivo experiments, mice (postnatal day 5) were injected intraneopallially with ibotenate acting on NMDA and metabotropic receptors, or S-bromowillardiine acting on AMPA-kainate receptors to produce excitotoxic stress and brain lesions. Systemically administered recombinant PRDX5 provided protection against ibotenate-induced excitotoxic stress. Brain lesions of animals given ibotenate and PRDX5 were up to 63% smaller than that given ibotenate alone. However, PRDX5 provided no prevention from lesions induced with S-bromowillardiine. A mutated recombinant PRDX5 that is devoid of peroxidase activity was also tested and showed no protection against lesions induced by either ibotenate or S-bromowillardiine. Two classical antioxidants, N-acetylcysteine and catalase-PEG, provided the same neuroprotective effect as PRDX5. For in vitro experiments, neocortical neurons were exposed to 300 microM NMDA alone, NMDA plus recombinant PRDX5, or NMDA, recombinant PRDX5 and dithiothreitol, a classical electron donor for peroxiredoxins. Recombinant PRDX5 plus dithiothreitol displayed a synergistic neuroprotective effect on NMDA-induced neuronal death. These findings indicate that reactive oxygen species production participates in the formation of NMDA receptor-mediated brain lesions in newborn mice and that antioxidant compounds, such as PRDX5, provide some neuroprotection in these models.