Hyperhomocysteinemia is a major and independent risk factor for vascular disease. Oxidative stress is a possible mechanism for homocysteine (Hcy)-induced endothelial dysfunction. Herein, we evaluated the antioxidant property of melatonin (MLT) in relation to the vasoconstrictive effect of Hey on the human umbilical artery. In an initial experiment in a cell-free system, a micromolar concentration of iron was found to catalyze oxygen-dependent oxidation of Hey. MLT (10 or 100 muM) did not affect oxygen-dependent oxidation of Hey. Next, smooth muscle contraction induced by prostaglandin F-2 alpha (10 muM) was measured in arterial strips. Hcy (10 to 500 muM) increased this vascular tension in a concentration-dependent manner (P < 0.0001). Addition of Fe2+ (10 M) significantly potentiated the Hey effect. Removal of endothelium (P < 0.05), pretreatment with a nitric oxide (NO) synthesis inhibitor (L-N-G-monomethylarginine, 200 M, P < 0.001), or pretreatment with a hydroxyl radical (OH) scavenger (mannitol, 10 mM, P < 0.001) significantly attenuated contraction potentiated by Hey plus Feet. At a much lower concentration than mannitol, MLT (1 to 100 M) significantly reduced the contractile effect of Hey and Fe2+ in a concentration-dependent manner. Hey plus Fe2+ significantly impaired calcium ionophore A 23187-induced relaxation (P < 0.0001), while MLT restored this relaxation in a concentration-dependent manner. These findings suggest that Hey potentiates vascular tension in human umbilical artery, possibly by suppressing bioavailable NO. MLT protects against the vasoconstrictive effect of Hey, most likely by scavenging OH arising from Hey autooxidation.