Susceptibility to drug-induced coronary vasospasm in rhesus monkeys increases after removal of the ovaries and can be normalized by adding back physiological levels of estradiol-17ss (E2) and/or natural progesterone (P) in vivo as reported recently by our group. Furthermore, the reactivity status (Ca2+ and protein kinase C responses) of freshly isolated and primary culture coronary artery vascular muscle cells (VMC) mimic the intact coronary artery responses to 5-HT + U46619. Since coronary reactivity is maintained in the isolated VMC, we hypothesized that the reactivity state inherent in the VMC was modulated directly by ovarian steroids in vitro as in the whole animal. To test this hypothesis, we treated hyperreactive VMC from ovariectomized (ovx) monkeys in vitro with E2 or P and measured VMC reactivity to combined stimulation with 5-HT and U46619, as determined by the amplitude and especially the duration of intracellular Ca2+ signals, as well as protein kinase C (PKC) activation/translocation. VMC were treated for 12 96 h with 3 100 pg/ml E2 (10 365 pM) and/or 0.3 3 ng/ml P (0.95 9.5 nM). Hyperreactive responses to the combination of 5-HT and U46619 in untreated VMC were significantly and dose-dependently reduced by treatment in vitro with physiological levels of either E2 or P for at least 24 h. Both the early transient and late sustained increases in intracellular Ca2+ and PKC translocation were blunted, and the effects of 0.2 nM E2 and 3.2 nM P were specifically antagonized by the receptor blockers ICI 182,780 (200 nM) and RU486 (15 nM), respectively. Antibodies to the estrogen receptor and progesterone receptor labeled nuclei in VMC, which were also positively labeled by a smooth muscle myosin heavy chain monoclonal antibody. These data indicate that natural ovarian steroids directly reduce hyperreactive 5-HT and thromboxane A2-stimulated Ca2+ and PKC responses of coronary artery VMC from surgically menopausal rhesus macaques. We hypothesize that vascular hyperreactivity, which may be a critical factor involved in the increased incidence of coronary artery vasospasm and ischemic heart disease in postmenopausal women, can be normalized by E2 and/or P through direct actions on coronary artery vascular muscle cells.