Ovariectomized (Ovx) rats were treated (s.c.) with estradiol benzoate (E2B) for 7 days, and then 10, 25 or 100 ng LH-RH were microinjected into the 3rd ventricle. Intraventricular LH-RH elevated plasma LH at 10 and 30 min in a dose-response manner. In experiment 2, Ovx rats were treated with E2B or oil for 7 days and then intraventricularly injected with 25 ng LH-RH. Two days later, 100 ng LH-RH were systemically administered. E2B treatment resulted in a greater release of LH in response to the intraventricular administration of LH-RH but not to the systemic injection. A direct measure of median eminence (ME) uptake of LH-RH was used in experiment 3. Ovx-E2B or oil-treated rats were decapitated 10 min after intraventricular injection of 125I-LH-RH, 125I or 3H-glycine. The ME region of the hypothalamus, cortex, anterior pituitary (AP) and plasma were solubilized and their radioactivity determined. E2B increased the radioactivity in the ME following injection of 125I-LH-RH but not in other tissues. Tissue uptake of 125I and 3H-glycine were similar in E2B or oil-treated rats. These data indicate E2B facilitates the incorporation of LH-RH into the ME from the cerebrospinal fluid (CSF).