Maintenance of pregnancy in the ewe depends on progesterone synthesized by the ovaries during the first third of gestation and by the placenta during the last two-thirds of gestation. There have been few investigations of in vitro synthesis of progesterone by the ovine placenta or of mechanisms controlling placental steroidogenesis. Weekly blood samples were collected from ten gestating ewes that were sacrificed for collection of placental tissue on either day 80 (n=5) or day 115 (n=5) of gestation. All blood samples were analyzed for progesterone by radioimmunoassay. Immediately after each ewe was sacrificed, cotyledons were collected from different areas of each placenta, minced, and incubated in 2 ml of culture medium for either .5, 1, or 2 h. Minced tissue (100 mg) was incubated either in the presence or absence of pregnenolone and the tissue was subject to addition of either human Chorionic Gonadotropin (hCG), 3’, 5', cyclic adenosine monophosphate (cAMP), luteinizing hormone-releasing hormone (LHRH) or no additional treatment. Progesterone concentration in the medium after incubation were determined by radioimmunoassay. The temporal pattern of in vivo progesterone synthesized during gestation exhibited a biphasic increase with elevations occurring between days 70 and 80, and 95 and 115 of gestation. The difference in progesterone concentration in incubation medium after incubation of placental tissue collected on day 80 or day 115 of gestation was similar in magnitude to the difference in plasma progesterone concentrations of samples collected on those days. At day 80 and 115 of gestation, in vitro progesterone concentrations were 8.4 and 15.3 ng/ml, respectively, while in vivo progesterone concentrations at day 80 and 115 were 7.2 and 12.5 ng/ml, respectively. Progesterone accumulation in the medium of placental tissue collected at 115 days of gestation was greater than concentrations of progesterone in tissue collected at 80 days of gestation (P<.01). HCG had no effect on progesterone accumulation in the incubation medium; however, cAMP and LHRH significantly affected progesterone concentrations when pregnenolone was added to the medium of tissue collected on day 115. Cyclic AMP significantly increased progesterone concentrations in the medium after 2 h of incubation over that of tissue incubated alone (354.1 vs 402.6 ng/g). Mean progesterone concentrations in the medium of tissue incubated with LHRH were lower than control incubations after 1 h (288.8 vs 355.6 ng/g) and 2 h (306.1 vs 354.1 ng/g) of incubation. / M.S.