The effect of progesterone on oxytocin-induced secretion of prostaglandin (PG) F_2α from bovine endometrial tissue explants was examined. Endometrial tissue from the late luteal phase were preincubated for 20 h in control medium. Explants were then treated for 6 h with control medium, oxytocin (10⁻⁷ M), progesterone (10⁻⁵ M), or both hormones. Oxytocin increased the medium concentration of 13,14-dihydro-15-keto-PGF_2α, whereas progesterone completely suppressed the stimulatory effect of oxytocin. In experiment 2, isolated endometrial epithelial cells were incubated with progesterone (10⁻⁵ M), oxytocin (10⁻⁷ M), and combinations of these hormones with or without actinomycin D (1 ng/ml). Only oxytocin stimulated secretion of PGF_2α, and this response was suppressed by progesterone. Oxytocin induced a rapid increase in intracellular concentrations of Ca² detected within 1 min of exposure of epithelial cells from the same cows. Progesterone pretreatment diminished this response. In experiment 3, direct effects of progesterone (2 nM–20 μM) on binding of ³H-oxytocin to the membrane preparation from epithelial cells were determined by saturation analysis. Oxytocin binding was suppressed by progesterone at every dosage tested. Progesterone is capable of suppressing the ability of oxytocin to induce endometrial secretion of PGF_2α. This effect appears to be mediated through a direct interference in the interaction of oxytocin with its own receptor.