Acute actions of estradiol and progesterone on synthesis and secretion of gonadotropins without the confounding effects of endogenous steroids and GnRH were examined by using the ovariectomized (OVX), hypothalamic-pituitary-disconnected (HPD) ewe. Thirty-two OVX ewes were subjected to HPD and randomly assigned to eight groups (4 ewes/group). An additional four OVX awes served as controls, Each of 12 OVX-HPD ewes received 4 s.c. implants of estradiol 24 h after HPD (implantation was designated as Time 0 h). Sixteen OVX-HPD ewes received no treatment and served as contemporary controls. The remaining four OVX-HPD ewes received implants of estradiol for 48 h and were administered 16 mg progesterone i.m. twice daily in the last 24 h of estradiol treatment. Blood samples and pituitary glands were collected from OVX-HPD control and steroid-treated ewes at 0, 12, 24, and 48 h. Amounts of gonadotropin subunit mRNAs and serum concentrations of gonadotropins were decreased at 24 h after HPD and remained unchanged thereafter in OVX-HPD controls. Although treatment with estradiol decreased pituitary content of LH and steady-state levels of mRNA for LH beta-subunit (p < 0.05) compared to contemporary controls, serum concentrations of LH were not affected. Serum concentrations and pituitary content of FSH decreased (p < 0.05) 24 h after initiating treatment with estradiol, but steady-state levels of FSH beta-subunit mRNA were unchanged. There was a transient decrease in pituitary content of FSH at 24 h of estradiol treatment. Steady-state levels of mRNA for alpha-subunit were unaffected by estradiol. Administration of progesterone did not influence the amount of mRNA for LH beta-subunit but increased pituitary content of LH (p < 0.05). Moreover, the amount of mRNA for FSH beta-subunit decreased (p < 0.05) in progesterone-treated ewes but pituitary content of FSH increased. There was a modest reduction in the amount of mRNA for a-subunit after administration of progesterone, but serum concentrations of gonadotropins were not affected. Collectively, from these data we conclude that estradiol and progesterone can act directly at the pituitary gland in the ewe to regulate steady-state levels of gonadotropin subunit mRNAs independent of GnRH.