The metabolism of GnRH by membrane-bound and serum-degrading enzymes may play an important role in the regulation of pituitary gonadotropin secretion. We examined GnRH metabolism by pituitary cells in vitro and the metabolism of exogenously administered GnRH in vivo in the presence and absence of estradiol (E2) and progesterone (P4). Ovariectomized cynomolgus monkeys were implanted with E2- and/or P4-filled Silastic capsules to simulate the estrogen and progesterone patterns of the normal menstrual cycle. Peripheral levels of GnRH after a 1-microgram iv injection were highest in the E2-replaced monkeys. Peripheral GnRH levels reached a higher peak and remained in circulation longer in monkeys treated with E2-filled Silastic implants than in those treated with E2 plus P4 or nonsteroid-replaced ovariectomized monkeys. In agreement with the in vivo data, GnRH was rapidly metabolized by acutely dispersed cells isolated from pituitaries removed from nonsteroid-replaced ovariectomized monkeys. Priming with E2 followed by P4 in vivo attenuated the clearance of GnRH in vitro, and E2 treatment alone almost completely blocked the ability of pituitary cells to bind and/or degrade GnRH in vitro. In a parallel study, cells prepared from rat pituitaries removed on the morning of proestrus (when serum E2 is highest) metabolized GnRH in vitro more slowly than pituitary cells removed at estrus, diestrus, or metestrus. In summary, our data suggest that E2 inhibits GnRH metabolism by monkey and rat pituitary cells in vitro and exogenously administered GnRH in vivo. Although the precise mechanism of action of E2 is unknown, inhibition of membrane-bound and serum proteases seems likely. The action of E2 may be to increase GnRH presentation to the pituitary and enhance LH and FSH secretion under conditions where circulating levels of the hormone are elevated, such as at midcycle.