In the present study, we evaluated the effect of melatonin, a well-known free radical scavenger and neuroprotector, against rotenone-induced oxidative stress in a hemiparkinsonian rat model. The effect of melatonin on glutathione (GSH) depletion caused by unilateral, intranigral infusion of rotenone was investigated employing a spectrofluorimetric procedure. We also studied the effect of melatonin on rotenone-induced changes in the antioxidant enzymes superoxide dismutase (SOD) and catalase in the cytosolic fractions of substantia nigra (SN), employing spectrophotometric procedures. Rotenone-induced hydroxyl radicals (*OH) in the isolated mitochondria, as measured employing a sensitive HPLC-electrochemical method, were significantly scavenged by melatonin. Melatonin treatment restored the rotenone-induced decrease in GSH level and changes in antioxidant enzyme (SOD and catalase) activities in the SN. Our results strongly indicate melatonin's beneficial use in Parkinson's disease therapy as an antioxidant.