A current hypothesis of ovine parturition proposes that fetal adrenal cortisol induces placental E2 production, which, in turn, triggers intrauterine PG production. However, recent evidence suggests that cortisol may directly increase PG production in trophoblast-derived tissues. To separate cortisol-dependent and estrogen-dependent PG production in sheep intrauterine tissues, we infused singleton, chronically catheterized fetuses beginning on day 125 of gestation (term, 147-150 days) with 1) cortisol (1.35 mg/h; n = 5); 2) cortisol and 4-hydroxyandrostendione, a P450aromatase inhibitor (4-OHA: 1.44 mg/h; n = 5); 3) saline (n = 5); or 4) saline and 4-OHA (n = 5). Fetal and maternal arterial blood samples were collected at 12-h intervals starting 24 h before infusion and continuing during treatment for 80 h or until active labor. Uterine contractility was measured by electromyogram recording of myometrial activity. Plasma E2, progesterone (P4), PGE2, and 13,14-dihydro- 15-keto-PGF2alpha were quantified by RIA. PGHS-II messenger RNA (mRNA) and protein expression were determined by in situ hybridization and Western blot analysis, respectively. Data were analyzed by ANOVA (P < or = 0.05). Labor-type uterine contractions were present after 68 h of cortisol infusion and had increased significantly by 80 h. Labor-type uterine contractions were induced after 68 h of cortisol plus 4-OHA infusion, but the contraction frequency remained less than that in the cortisol-treated animals. Fetal cortisol infusion increased fetal and maternal plasma E2 concentrations and decreased the maternal plasma P4 concentration significantly; concurrent 4-OHA infusion attenuated the increase in fetal and maternal plasma E2, but not the decrease in maternal plasma P4. The fetal plasma PGE2 concentration increased after both cortisol and cortisol plus 4-OHA infusion. The maternal plasma 13,14-dihydro-15-keto-PGF2alpha concentration rose after fetal cortisol infusion, but not after cortisol plus 4-OHA infusion. Placental trophoblast PGHS-II mRNA and protein expression were increased significantly after both cortisol and cortisol plus 4-OHA infusion. Endometrial PGHS-II mRNA and protein expression increased after cortisol infusion, but not after cortisol plus 4-OHA infusion. Plasma steroid and PG concentrations, uterine activity pattern, and intrauterine PGHS-II expression were not altered in either control group. We conclude that these data suggest distinct pathways of intrauterine PG synthesis: a cortisol-dependent/E2-independent mechanism within trophoblast tissue leading to elevations in fetal plasma PGE2, and an E2-dependent mechanism within maternal endometrium that leads to increased maternal plasma PGF2alpha and appears necessary for uterine activity and parturition.