Many in vitro studies show estrogen regulation of the hypothalamic pro-opiomelanocortin (POMC) system, including a decrease in hypothalamic POMC mRNA after estradiol treatment. Because such in vivo experiments do not allow one to determine whether peripheral, interacting systems or extra-hypothalamic brain regions are involved in this regulation, we sought to establish whether estrogen acts directly in hypothalamus to decrease POMC mRNA. Using an in vitro approach, we studied effects of estradiol (E2) on POMC/cyclophilin mRNA concentrations (RNAse protection assays) in neuronal cultures derived from day 17 fetal rat hypothalamus. Chemically defined medium was deprived of progesterone for 2 days prior to E2 treatment and for the duration of the study. E2 (10(-13)-10(-9) M) dose-dependently decreased POMC mRNA concentrations during a 2-day treatment. Whereas the lowest dose (10(-13) M) of E2 resulted in a statistically significant 44% decrease in POMC mRNA concentrations relative to control cultures, this inhibitory effect was lost because higher doses (10(-11) and 10(-9) M) did not produce statistically significant decrements (22 and 16%, respectively) in POMC mRNA concentrations. Additional time course studies revealed that this decrease in POMC mRNA can be seen as early as 4 h after E2 (10(-13) M) treatment. We conclude that E2 inhibition of POMC mRNA concentrations in hypothalamic neuronal cultures indicates that this inhibition can occur directly in hypothalamus.