In vitro extracellular Mg²⁺ concentration ([Mg²⁺]₀) produces endothelium-dependent and endothelium-independent relaxations in rat aorta in a concentration-dependent manner. These relaxant effects of Mg²⁺ on intact rat aortic rings, but not denuded rat aortic rings, were suppressed by either N^G-monomethyl-L-arginine (L-NMMA), N-nitro-L-arginine methyl ester (L-NAME), or methylene blue. The inhibitory effects of L-NMMA and L-NAME could be reversed partly by L-arginine. [Mg²⁺]₀-induced dilatation in vivo in rat mesenteric arterioles and venules was almost completely inhibited by N^G-nitro-L-arginine and L-NMMA. Removal of extracellular Ca²⁺ concentration ([Ca²⁺]₀) or buffering intracellular Ca²⁺ concentration in endothelial cells, with 10 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, markedly attenuated the relaxant effects of Mg²⁺. Mg²⁺ produced nitric oxide (NO) release from the intact aortic rings in a concentration-dependent manner. Removal of [Ca²⁺]₀ diminished the increased NO release induced by elevated levels of [Mg²⁺]₀. In vivo infusion of increasing doses (1-30 µM/min) of MgSO₄, directly into the femoral veins of anesthetized rats, elicited significant concentration-dependent sustained increases in serum total Mg and concomitant decreases in arterial blood pressure. Before and after employment of various doses of MgSO₄, intravenous administration of either L-NMMA (10 mg/kg) or L-NAME (10 mg/kg) increased (i.e., reversed) the MgSO₄-lowered blood pressure markedly, and intravenous injection of L-arginine restored partially the increased blood pressure effects of both L-NMMA and L-NAME. Our results suggest that 1) small blood vessels are very dependent on NO release for Mg²⁺ dilatations and 2) the endothelium-dependent relaxation induced by extracellular Mg²⁺ is mediated by release of endothelium-derived relaxing factor-NO from the endothelium, and requires Ca²⁺ and formation of guanosine 3',5'-cyclic monophosphate.