Sensitivity of neurons to estrogen in down-regulation of estrogen receptor alpha (ERalpha) can be thought to make a sex difference in regulatory system of reproductive activities. In this study, to investigate the sex difference of expression of ERalpha in the hypothalamus and midbrain, the number of ERalpha immunoreactive (-ir) cells was counted in orchidectomized (OCX) and ovariectomized (OVX) rats with or without treatment with estrogen. A week after the gonadectomy, 5 rats in each female and male were injected with 1mg estradiol benzoate (EB). The remaining 5 rats in both sexes did not receive EB. The brain was fixed 24h after EB-injection and 50 microm-serial frozen sections were made. After immunohistochemical staining for ERalpha, the number of ERalpha-ir cells was counted in a 0.2-mm2 frame in the anteroventral periventricular nucleus (AVPvN), the ventrolateral part of the ventromedial hypothalamic nucleus (vlVMN), the arcuate nucleus (ARCN), and the lateral mesencephalic central gray (lMCG) in 2 or 3 sections. The total number of ERalpha-ir cells was changed to a density value (number per 1mm3). As the results, in EB-treated rats, the density of ERalpha-ir cells in all regions, except the male AVPvN and male lMCG, were lower than those in untreated rats of both sexes. In the vlVMN, the density of ERalpha-ir cells in OVX rats was higher than in OCX rats. These results suggest that there are sex and regional differences in the mechanisms of down-regulation of ERalpha by estrogen in the rat brain.