Estrogen and glucocorticoids interact in multiple aspects of endocrine regulation by exerting opposing influences on the expression of selective genes. In rats, ER- is the predominant form of ER present in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, suggesting its involvement in neuroendocrine regulation. To date, the hormonal regulatory profile of the ER- gene in the rat central nervous system has not been closely elucidated. In the present study, we first examined the effects of dexamethasone (DEX) and estradiol benzoate (EB) on the ER- protein expression in the PVN and SON of ovariectomized female rats. In the SON and the parvocellular and magnocellular parts of the PVN, the number of ER- immunoreactive nuclei significantly increased following DEX treatment compared with the control group, whereas EB treatment caused a significant decrease. The effect of EB was consistent across other brain nuclei such as the anteroventral periventricular nucleus and medial preoptic nucleus. To determine the molecular level at which DEX and EB control ER- expression, we examined the effects of these steroids on ER- mRNA levels using real-time RT-PCR. EB significantly decreased the expression of ER- mRNA in the PVN (P = 0.0006) and SON (P < 0.01). In contrast, DEX did not change ER- mRNA levels. These results indicate that glucocorticoids and estrogen exert opposing regulatory influences on the ER- gene expression. This may represent a mechanism by which these steroids can alter the cellular sensitivity of ER- expressing neurons to subsequent steroidal activation.