The influence of melatonin on alpha- and beta-adrenergic responses of vascular smooth muscle ex vivo were examined in these experiments. Melatonin caused a dose-dependent relaxation of precontracted (30 mM KCl) vascular smooth muscle. This response was not affected by the removal of vascular endothelium. Melatonin (10 nM) reduced the efficacy, but not potency, of the contractile responses to methoxamine and clonidine. Melatonin had no effect on the vascular beta-adrenergic response to isoproterenol. Pretreatment of vascular rings with lithium sulfate (0.1 mM) completely blocked the relaxation in response to melatonin, suggesting that the inositol phosphate pathway may be involved in this relaxation. Furthermore, pretreatment of vascular rings with phorbol 12-myristate-13-acetate, an activator of protein kinase C, antagonized the relaxation response to melatonin. Pharmacologic doses of melatonin (0.1 mM) slightly impaired the vascular smooth muscle response to calcium. These data indicate that melatonin per se is capable of relaxing vascular smooth muscle and that low doses of melatonin impair alpha-1 and alpha-2 adrenergic responses without changes in the beta adrenergic response of vascular smooth muscle.