Hypothalamic gonadotropin releasing hormone (GnRH) and its analogs have been shown to regulate progestin production by ovarian granulosa cells treated with FSH and prolactin. The present study examines the direct modulation of an ovarian progesterone metabolizing enzyme by GnRH and its analogs in vitro and in vivo. Granulosa cells from hypophyseclomized, diethylstilbestrol-treated rats were cultured with GnRH or its analogs. After 2 days, cells were homogenized and 20α-hydroxysteroid dehydrogenase (20α-OH-SDH) activity was measured using a direct enzyme assay. GnRH stimulated 20α-OH-SDH activity in a dose-dependent manner with an ED 50 of 8.95 × 10 −9M and a GnRH aganist ([des-Gly 10, d-Lcu 6, (N 2Me)Leu 7,Pro 9NHEt]GnRH) was shown to be 5.6 fold more potent than GnRH. The stimulatory effect of GnRH was inhibited by concomitant treatment with a GnRH antagonist ([ d-pGlu 1, d-Phe 2 d-Trp 3,6]GnRH) with a half maximal inhibitory dose ratio of 3 1 ([antagonist]/[GnRH]). Time course studies indicated that 12 h of GnRH treatment were required to stimulate a measurable increase in enzyme activity. Cycloheximide treatment inhibited the GnRH action, suggesting that GnRH induces the synthesis of 20α-OH-SDH. Furthermore, treatment with GnRH and its analogs in hypophysectomizcd rats in vivo stimulated enzyme activity without affecting the K m of the enzyme, whereas the stimulatory effect of GnRH was blocked by concomitant treatment with a potent GnRH antagonist. These studies demonstrated that GnRH and its analogs may directly regulate the synthesis of ovarian 20α-OH-SDH in vitro and in vivo.