Background/Aims: The contributions of the three principal ovarian steroid hormones (estradiol, progesterone and testosterone) to the regulation of estrogen receptor alpha (ERα) levels in the rat brain were examined during the estrous cycle. Methods: Receptor concentrations were measured using an in vitro autoradiographic technique designed to separately quantify free, unoccupied receptors and receptors ‘occupied' by (bound to) endogenous hormone. Results: ERα occupation increased at proestrus and declined at estrus, reflecting changes in circulating estradiol and testosterone levels. Total ERα content followed a pattern that was the inverse of the occupation data, falling over the night of proestrus. Between 2.00 and 10.00 a.m. on the day of estrus, total ERα concentrations recovered in all brain regions except the ventromedial nucleus (VMN), in which ERα binding remained depressed at estrus. Administration of the progesterone antagonist mifepristone on the afternoon of proestrus resulted in recovery of ERα levels in the VMN by the morning of estrus, consistent with the hypothesis that the preovulatory progesterone surge selectively inhibits VMN ERα expression. Residual ERα occupation observed at estrus, when estradiol is not detectable in the serum, likely reflects intracranial aromatization of circulating androgens, since the pattern of receptor occupation observed at this stage of the cycle could be reproduced in ovariectomized rats by replacement with testosterone. Conclusion: These findings indicate that ERα binding in the brain fluctuates during the rat estrous cycle in a region-specific manner and suggest that local aromatization of testosterone may contribute significantly to ERα occupation when circulating estradiol levels are low.