Acute estrogen administration relaxes vascular smooth muscle by decreasing intracellular Ca2+ concentration ([Ca2+]i). In the present study, we examined the hypothesis that this reduction in [Ca2+]i is mediated in part by enhanced Ca2+ efflux. Coronary artery smooth muscle cells were isolated from gonad-intact, sexually mature female pigs. The [Ca2+]i response to endothelin-1 was measured using fluo 3 and confocal microscopy. 17beta-Estradiol (E2beta), but not 17alpha-estradiol or triamcinolone acetonide, caused a concentration-dependent (IC50 = 10 nM) decrease in the [Ca2+]i response to endothelin-1. This decrease was blocked by the specific estrogen receptor antagonist ICI-182780. Under conditions in which Ca2+ influx and sarcoplasmic reticulum Ca2+ reuptake were blocked, E2beta still decreased [Ca2+]i. The response was blocked by extracellular lanthanum. These data indicate that E2beta decreases [Ca2+]i in coronary artery smooth muscle by affecting Ca2+ efflux via a receptor-mediated mechanism.