Experiments were conducted to characterize the nongenomic effects of progesterone (P4) on binding of oxytocin (OT) to its receptor and signal transduction in the ovine endometrium. The dose-response relationship of P4 to OT binding was examined. Membranes from endometrial tissue of ovariectomized hormone-treated ewes were preincubated in the presence of P4 for 1 h followed by OTR analysis. Progesterone interfered with the binding of OT in a dose-dependent manner. Endometrium was then recovered from cyclic ewes and divided into explants. Treatment consisted of two dosages of P4 and two dosages of OT. Explants were analyzed for total inositol mono- (IP), bis- (IP₂), and trisphosphate (IP₃) content. Preincubation with progesterone for 10 min significantly interfered with OT stimulation of IP₂ and IP₃ synthesis. Oxytocin increased IP production, but there was no detectable effect of P4. In the next experiment endometrial explants were cultured in the absence or the presence of arachidonic acid (AA). Explants were then exposed for 1 h to medium containing vehicle or P4. After incubation explants were challenged with OT and the media collected and analyzed for PGFM by RIA. Treatment of explants with AA increased PGF₂ content compared with that of controls. Brief exposure to P4 significantly decreased OT-induced PGF₂ secretion from explants previously exposed to medium or AA. Collectively, these data are interpreted to indicate that the observed reduction in OT-induced IP₂ and IP₃ production and OT-induced PGF₂ secretion was due to P4 inhibition of OT binding to its receptor.