In a study of cellular mechanisms of progesterone's antiestrogenic action on behavior and neuroendocrine responses, we investigated the influence of progesterone on the concentration of estrogen receptors in the hypothalamus-preoptic area (HP), anterior pituitary gland (AP), and uterus of chronically estradiol-treated ovariectomized rats. Ovariectomized (OVX) rats were implanted s.c. with 15 mm silastic capsules of estradiol. One week later, they were injected with progesterone or oil vehicle and killed 6 h or 24 h later. Confirming previous reports, progesterone caused a decrease in the concentration of uterine cytosol and nuclear estrogen receptors at both times. Less consistent results were obtained in HP and AP; a decrease in the concentration of HP cytosol estrogen receptors was detected at 6 h, as was a small decrease in the concentration of HP nuclear estrogen receptors at 24 h. More consistent results were seen when a low priming dose of estradiol was used. Although progesterone was without effect on the concentration of nuclear estrogen receptors in HP and AP at 6 h, cytosol receptor levels were depressed by 25% in HP and 14% in AP. At 24 h after progesterone injection, nuclear estrogen receptor levels were decreased in all tissues, while cytosol estrogen receptor levels remained depressed. A study of the time course of progesterone's suppression of cytosol estrogen receptor concentration revealed that the effect is transient, occurring by 6 h after progesterone injection, but returning to baseline by 48 h after injection. Scatchard analysis confirmed that the decreased concentration of cytosol binding in HP was due to a decrease in the concentration of binding sites. As with nearly all of progesterone's neuroendocrine effects, the suppression of estrogen receptor levels requires estrogen priming. HP and AP cytosol from progesterone-treated rats did not seem to contain an estrogen receptor-regulatory factor as do uterine cell nuclei; loss of binding sites at 37 degrees C was no faster in cytosol from progesterone-treated rats. These results demonstrate that, under some conditions, progesterone decreases HP and AP estrogen receptor concentrations. Unlike progesterone's action in the uterus, the primary effect in the brain and pituitary gland seems to be on the cytosol receptor.