This study investigated whether the induction of inducible nitric oxide synthase (iNOS) produced by acute exercise in rat skeletal muscle could be prevented by melatonin and whether iNOS down-regulation was related to inhibition of nuclear factor kappaB (NF-κB) activation. Male Wistar rats received melatonin i.p. at a dose of 1.0 mg/kg body weight 30 min before being exercised for 60 min on a treadmill at a speed of 25 m/min and a 10% slope. Exercise caused a significant induction of iNOS protein levels and a marked activation of NF-κB that were significantly prevented in rats treated with melatonin. Exercise also resulted in increased IκB kinaseα (IKKα) and phosphorylated IκBα protein levels, whereas IκBα content decreased. These effects were blocked by melatonin administration. The increase in the muscle concentration of thiobarbituric acid reactive substances and in the oxidized/reduced glutathione ratio induced by exercise was partially prevented by melatonin. Our data indicate that melatonin has potent protective effects against damage caused by acute exercise in rat muscle, preventing oxidative stress, NF-κB activation and iNOS over-expression. These findings support the view that melatonin treatment, by abolishing the IKK/NF-κB signal transduction pathway, might block the production of noxious mediators involved in the inflammatory process.