In this study, the protective effect of melatonin against kainate (KA)-induced neurotoxicity was evaluated in vitro and in vivo. In rat brain synaptosomes, KA-induced oxidative stress was measured as shown by significant increases in both the basal generation of reactive oxygen species (ROS), assessed by a fluorescent method, and lipid peroxidation, evaluated as malondialdehyde (MDA) levels. Melatonin decreased, in a concentration-dependent manner, KA-induced lipid peroxidation. The intrinsic fluorescence of melatonin molecule hindered the evaluation of its protective effect against KA-induced ROS generation. However, melatonin was able to reduce FeSO4/ascorbate-induced ROS generation. The melatonin protective effect was confirmed by in vivo experiments: 73% of rats injected with KA (10 mg/kg i.p.) died within 5 days; melatonin administration i.p. significantly reduced mortality of the animals. The present results suggest that melatonin might be considered a pharmacological agent for the treatment of neurodegenerative pathologies.