The role of melatonin in improving mitochondrial respiratory chain activity and increasing ATP production in different experimental conditions has been widely reported. To date, however, the mechanism(s) involved are largely unknown. Using high-resolution respirometry, fluorometry and spectrophotometry we studied the effects of melatonin on normal mitochondrial functions. Mitochondria were recovered from mouse liver cells and incubated in vitro with melatonin at concentrations ranging from 1 nm to 1 mm. Melatonin decreased oxygen consumption concomitantly with its concentration, inhibited any increase in oxygen flux in the presence of an excess of ADP, reduced the membrane potential, and consequently inhibited the production of superoxide anion and hydrogen peroxide. At the same time it maintained the efficiency of oxidative phosphorylation and ATP synthesis while increasing the activity of the respiratory complexes (mainly complexes I, III, and IV). The effects of melatonin appeared to be due to its presence within the mitochondria, since kinetic experiments clearly showed its incorporation into these organelles. Our results support the hypothesis that melatonin, together with hormones such as triiodothyronine, participates in the physiological regulation of mitochondrial homeostasis.