Melatonin is involved in the regulation of the cardiovascular system through modulation of sympathetic function and the nitric oxide (NO)-related pathway and interaction with MT1/MT2 receptors. However, information regarding its direct actions on coronary blood flow and cardiac function is scarce. The present study therefore determined the primary in vivo effect of melatonin on cardiac function and perfusion and the involvement of the autonomic nervous system, MT1/MT2 receptors and NO. In 35 pigs, melatonin infused into the coronary artery at 70 pg for each ml/min of coronary blood flow while preventing changes in heart rate and arterial pressure increased coronary blood flow, dP/dtmax, segmental shortening and cardiac output by about 12%, 14%, 8% and 23% of control values (P<0.05). These effects were accompanied by an increase of coronary NO release of about 46% (P<0.05) of control values. The above responses were graded in a further 5 pigs. Moreover, the blockade of muscarinic cholinoreceptors (n=5) and of a-adrenoreceptors (n=5) did not abolish the observed responses to melatonin. After b1-adrenoreceptors blocking (n=5), melatonin failed to affect cardiac function whereas b2-adrenoreceptors (n=5) and NO-synthase inhibition (n=5) prevented the coronary response and the effect of melatonin on NO release. Finally, all effects were prevented by MT1/MT2 receptors inhibitors (n=10). In conclusion, melatonin primarily increased coronary blood flow and cardiac function through the involvement of MT1/MT2 receptors, b-adrenoreceptors and NO release. These findings add new information about the mechanisms through which melatonin physiologically modulates cardiovascular function and exerts cardioprotective effects.