Melatonin displays antioxidant and free radical scavenger properties. Due to its ability with which it enters cells, these protective effects are manifested in all subcellular compartments. Recent studies suggest a role for melatonin in mitochondrial metabolism. To study the effects of melatonin on this organelle we used ruthenium red to induce mitochondrial damage and oxidative stress. The results show that melatonin (10 mg/kg i.p.) can increase the activity of the mitochondrial respiratory complexes I and IV after its administration in vivo in a time-dependent manner; these changes correlate well with the half-life of the indole in plasma. Melatonin administration also prevented the decrease in the activity of complexes I and IV due to ruthenium red (60 μg/kg i.p.) administration. At this dose, ruthenium red did not induce lipid peroxidation but it significantly reduced the activity of the antioxidative enzyme glutathione peroxidase, an effect also counteracted by melatonin. These results suggest that melatonin modulates mitochondrial respiratory activity, an effect that may account for some of the protective properties of the indoleamine. The mitochondria-modulating role of melatonin may be of physiological significance since it seems that the indoleamine is concentrated into normal mitochondria. The data also support a pharmacological use of melatonin in drug-induced mitochondrial damage in vivo.