The available evidences suggest that vitamin D has cardiovascular effects besides regulating calcium homeostasis. To examine the effect of 1,25-dihydroxyvitamin D3, the major metabolite of vitamin D, on endothelium-dependent contractions, rings of spontaneously hypertensive rats (SHR) aorta were suspended in organ chambers for isometric force measurements. Rings incubated with N-nitro-L-arginine methyl ester (L-NAME) and then exposed to increasing concentrations of acetylcholine, ATP or the calcium ionophore, to trigger contractions. This was done in the absence or presence of 1,25-dihydroxyvitamin D3. The release of prostacyclin after acetylcholine or A23187 stimulation was also measured. The cytosolic-free calcium concentration was measured by confocal microscopy after incubation with the fluorescent dyes Fluo-4 and Fura-red. The presence of vitamin D receptors was confirmed using immunohistochemistry. Acetylcholine- and ATP-induced endothelium-dependent contractions were reduced significantly compared to those obtained in the absence of the drug. This effect was not present if A23187 was used as an agonist. The acetylcholine- but not the A23187-induced release of prostacyclin was reduced by the acute administration of 1,25-dihydroxyvitamin D3. Exposure to 1,25-dihydroxyvitamin D3 reduced the increase in cytosolic free calcium concentration caused by acetylcholine but not by A23187 in cells. Vitamin D receptors were densely distributed in the endothelium. Inecalcitol (19-nor-14-epi-23-yne-1,25-dihydroxyvitamin D3), a synthetic analog of vitamin D, caused a comparable depression of endothelium-dependent contractions as 1,25-dihydroxyvitamin D3. These results demonstrate that vitamin D3 modulates vascular tone by reducing calcium influx into the endothelial cells and hence decreasing the production of endothelium-derived contracting factors.