Bilaterally ovariectomized, nonpregnant female CD rats were studied to investigate the effect of estradiol treatment on in vitro myometrial responsiveness to oxytocin and prostaglandin F2alpha. The first study investigated dose-dependent effects. Seven days after ovariectomy rats were given a single s.c. dose of corn oil (n = 4) or estradiol (5 microg, n = 5; 15 microg, n = 5; 50 microg, n = 4). A second identical injection of corn oil or estradiol was administered 24 h after the initial injection. Rats were killed 48 h after the first injection. A second study investigated time-dependent effects of estradiol treatment. A second group of ovariectomized rats received s.c. estradiol (50 microg) seven days after ovariectomy. These rats were killed either 12 h (n = 5) or 24 h (n = 4) after injection. Full-thickness cross-sections of uteri were suspended in vitro in the longitudinal direction in a superfusion system. Cumulative concentration-response curves were constructed to oxytocin and prostaglandin F2alpha. Both the duration and dose of estradiol treatment significantly (p < 0.05) attenuated baseline contractile activity, and the maximum myometrial response to oxytocin and prostaglandin F2alpha. Estradiol, in a dose-dependent manner, significantly reduced myometrial sensitivity (p < 0.05) for oxytocin and prostaglandin F2alpha.