In isolated rat aorta, endothelin (ET)-3 increased cytosolic Ca²⁺ ([Ca²⁺]_i) in the endothelium at a concentration (100 nM) which had little effect on muscle resting tone. In the absence of external Ca²⁺, ET-3 still transiently increased endothelial [Ca²⁺]_i. Verapamil (10 μM) did not change the effects of ET-3. In aortas stimulated with 100 nM norepinephrine, 100 nM ET-3 relaxed the muscle with an increase in endothelial [Ca²⁺]_i. An inhibitor of nitric oxide synthase, 100 μM N^G-monomethyl-L-arginine, inhibited the relaxant effect of ET-3 but not the increase in endothelial [Ca²⁺]_i. In the absence of the endothelium or in the presence of an antagonist of ET_B receptors, 3 μM IRL 1038, the ET-3-induced increase in endothelial [Ca²⁺]_i and relaxation of norepinephrine-induced contraction were inhibited. Under these conditions, ET-3 increased smooth muscle [Ca²⁺]_i and induced contraction, both of which were inhibited by an inhibitor of ET_A receptors, 3 μM BQ123. These results suggest that ET-3 acts on ET_B receptors in the vascular endothelium to increase [Ca²⁺]_i by releasing Ca²⁺ from storage sites and by opening non-L type Ca²⁺ channels, activates nitric oxide synthase, releases nitric oxide and relaxes vascular smooth muscle. Although ET-3 also activates ET_A receptors in smooth muscle to induce contraction, this effect is overcome by the relaxant effect mediated by ET_B receptors.