Previous reports showed that 17-estradiol (E2) implants attenuate in vivo coronary hyperreactivity (CH), characterized by long duration vasoconstrictions (in coronary angiographic experiments), in menopausal rhesus monkeys. Prolonged Ca²⁺ contraction signals that correspond with CH in coronary vascular muscle cells (VMC) to the same dual constrictor stimulus, serotonin (S) plus the thromboxane analog U46619 (U), in estrogen deprived VMC were suppressed by >72 hr in E₂. The purpose of this study was to test whether an endogenous estrogen metabolite with estrogen receptor (ER) binding activity, estriol (E₃), suppresses in vivo and in vitro CH. Four-week treatment with E₃ in vivo significantly attenuated the angiographically evaluated vasoconstrictor response to intracoronary S+U challenge. In vitro treatment of rhesus coronary VMC for >72 hr with nanomolar E₃ attenuated late Ca²⁺ signals. This reduction of late Ca²⁺ signals also appeared after >72 hr treatment with subnanomolar 3Adiol (an endogenous dihydrotestosterone metabolite with ER binding activity). R,R-tetrahydrochrysene, a selective ER antagonist, significantly blocked the E_3- and 3Adiol-mediated attenuation of late Ca²⁺ signal increases. ER and thromboxane-prostanoid receptors (TPR) were co-expressed in coronary arteries and aorta. In vivo E₃ treatment attenuated aortic TPR expression. Furthermore, in vitro treatment with E₃ or 3Adiol down-regulated TPR expression in VMC, which was blocked for both agonists by pretreatment with R,R-THC. E_3- and 3Adiol-mediated reduction in persistent Ca²⁺ signals are associated with ER-mediated attenuation of TPR expression and may partly explain estrogen benefits in coronary vascular muscle.