The ability of estradiol to affect phenylephrine-induced contraction and the subsequent increase in resting tone (IRT), associated with capacitative Ca²⁺ entry across the plasma membrane, was evaluated in rat aortic rings incubated in Ca²⁺-free solution. The incubation with estradiol (1nM to 100 nM, 5 min) inhibited both; the phenylephrine-induced contraction and the IRT. Neither cycloheximide (1 µM; inhibitor of protein synthesis) nor tamoxifen (1 µM, blocker of estrogenic receptors) modified the effects of estradiol. Estradiol (100 µM) also blocked the contractile response to serotonin (10 µM) but not to caffeine (10 mM). In addition, estradiol (100 µM) inhibited the contractile responses to cyclopiazonic acid (1 µM; selective Ca²⁺-ATPase inhibitor) associated with capacitative Ca²⁺ influx through non-L-type Ca²⁺ channels. Finally, estradiol inhibited the Ca²⁺-induced increases in intracellular free Ca²⁺ (after pretreatment with phenylephrine) in cultured rat aorta smooth muscle cells incubated in Ca²⁺-free solution. In conclusion: Estradiol in a concentration-dependent manner, interfered with Ca²⁺-dependent contractile effects mediated by the stimuli of alpha1-adrenergic and serotonergic receptors and inhibited the capacitative Ca²⁺ influx through both L-type and non L-type Ca²⁺ channels. Such effects are in essence non-genomic and non-mediated by the intracellular estrogenic-receptor.