Abstract: This study compared the effects of melatonin supplementation on markers of oxidative stress, and on the activity and expression of antioxidant enzymes in the liver of young (3-month-old) and aging (24-month-old) rats. Animals were supplemented with melatonin in the drinking water (20 mg/L) for 4 wk. Liver concentration of thiobarbituric-reactive substances (TBARS), as an index of lipid peroxidation, and the oxidized to reduced glutathione ratio significantly increased in aged rats (+58%), while values did not significantly differ from the young in aged animals receiving melatonin. Significant decreases in the liver activities of Cu,Zn-superoxide dismutase (SOD) (−25%), cytosolic (−21%) and mitochondrial (−40%) glutathione peroxidase (GPx), and catalase (CAT) (−34%) were found in aged rats. Melatonin abolished these changes and also prevented the reduction of Cu,Zn-SOD (−33%), cytosolic GPx (−30%), and mitochondrial GPx (−47%) liver protein content as measured by Western blot. Reductions in Cu,Zn-SOD mRNA (−39%), and GPx mRNA (−86%) levels induced by aging were also abolished by melatonin. In summary, our data indicate that melatonin treatment abrogates oxidative stress in the liver of aged rats, and that prevention of the decreased activity of CAT and the downregulation of Cu,Zn-SOD and GPx gene expression contribute to this effect.