The objective of the present research was to determine, by use of the progesterone receptor antagonist RU-486, whether progesterone by autocrine/paracrine action suppresses phosphatidylinositol hydrolysis, and thus, the production of prostaglandin F2a during the midluteal phase of the estrous cycle in ewes. Two experiments were performed. Experiment 1 consisted of 10 ewes (day 8 of the estrous cycle) with an initial in vivo treatment of 10 ug RU-486 in saline or vehicle and blood samples collected before treatment and 10 min after for progesterone analysis. Treatment of RU-486 or vehicle was injected into the ovarian artery and corpora lutea were removed after 10 min of exposure. Fifty mg tissue slices were incubated with treatments of 3H myo-inositol (10 uCi) for 90 min followed by incubation (15 min) in the absence and presence of PGF2a (10 nM), to evaluate the effects of RU-486 and PGF2a on phosphoinositide hydrolysis. Total labeled inositol phosphates were recovered by use of column chromatography. Data were expressed as cpm of 3H myo-inositol/mg tissue. Analysis of variance revealed a significant PGF2a x RU-486 interaction (p <0.05) arising as a result of both agents being effective in stimulating incorporation of 3H myo-inositol into the inositol phosphates. Serum concentrations of progesterone were significantly reduced in both the control and RU-486 treated ewes (p<0.05). In a subsequent experiment, luteal tissue was collected on day 8 of the estrous cycle as in experiment 1. Fifty mg luteal slices were incubated with 3H myo-inositol (10 Ci) for 90 min and samples were again exposed to RU-486 (2 uM) in the absence and presence PGF2a (1 uM) for 15 min. Total labeled inositol phosphates were again recovered by use of column chromatography. Comparable to data of experiment 1, in vitro exposure of tissue to both RU-486 and PGF2a in experiment 2 caused an increase in incorporation of 3H myo-inositol into phosphoinositide phosphates (RU-486 x PGF2a interaction, p<0.05). Progesterone levels of the incubation medium demonstrated an increase in response of luteal tissue to RU-486 and was significantly increased by exposure to PGF2a in just 15 min (p<0.05). Collectively, these data suggest that progesterone may act nongenomically in an autocrine/paracrine manner to inhibit phosphatidylinositol hydrolysis, and by this action prevent production of PGF2a. Such inhibition of endogenous PGF2a production, by progesterone would prevent self-destruction of the CL during the midluteal phase of the cycle.