We have previously reported that melatonin modifies carbohydrate and lipid utilization in exercised rats, maintaining glycemia and reducing plasma and liver lactate and plasma β-hydroxybutyrate. This study was undertaken to determine whether effects on fuel metabolism were related to changes in nitric oxide (NO) production or growth hormone (GH) secretion. Male Wistar rats received melatonin i.p. at a dose of 0.5 mg/kg body weight 30 min before being exercised to exhaustion on a treadmill at a speed of 24 m/min and a 12% slope. Melatonin ameliorated the decrease in plasma glucose and the increase in plasma urea, free fatty acid, β-hydroxybutyrate, and nitrite induced by exercise. Melatonin-treated exercised rats had significantly elevated liver glycogen content and hepatic tissue showed a lowered expression of both inducible and constitutive NO synthase (iNOS and cNOS). Administration of the NO inhibitor NG-nitro-l-arginine (l-NAME) to exercised rats caused a significant reduction in plasma nitrite, but liver glycogen and biochemical parameters in blood did not significantly differ from untreated exercised animals, indicating the absence of a direct association between melatonin effects on fuel metabolism and NO levels. Although results of treatment with pyridostigmine, a cholinergic agonist drug that stimulates GH release, partially differed from that of melatonin, modulation of GH secretion could play a role in the metabolic actions of the hormone because effects of melatonin on exercised rats were almost completely blocked by simultaneous administration of l-NAME.