Objective:To test the hypothesis that deuterium oxide (D2O) might induce endothelium-dependent and nitric oxide-mediated vasodilation by altering intracellular calcium concentration ([Ca2+]¡) in vascular endothelial cells.Design:The endothelium-dependent and nitric oxide-mediated regulation of vascular tone by D2O was first examined in perfused rat mesenteric arterial beds. Direct effects of D2O on [Ca2+]¡in cultured vascular endothelial cells were then examined in order to elucidate a cellular mechanism for D2O-induced vasodilation.Methods:Vascular reactivity was assayed by measuring the perfusion pressure of isolated rat mesenteric arterial beds. [Ca2+]¡in cultured bovine and rat vascular endothelial cells was measured with the fura-2 fluorescence technique.Results:D2O relaxed precontracted rat mesenteric arterial beds with an intact endothelium. This effect of D2O was either inhibited by Nco-nitro-L-arginine (a nitric oxide synthase inhibitor) or eliminated by the removal of endothelium. In cultured bovine aortic, rat aortic, and rat tail artery endothelial cells, D2O induced a biphasic increase in [Ca2+]¡, with a characteristic initial transient increase followed by various patterns of sustained [Ca2+]¡increase. The sustained phase was entirely dependent on the extracellular calcium entry.Conclusions:A direct effect of D2O on [Ca2+]¡in vascular endothelial cells may be responsible for the endothelium-dependent, presumably nitric oxide-mediated, vasodilation induced by D2O in precontracted vessels. From these results new mechanisms can be explored for the antihypertensive effect of D2O and new avenues can be developed to study the functional integrity of the endothelium-dependent regulation of the vascular tone.