Progesterone (P) is one of several local mediators in the ovulatory cascade in the rat. The precise mechanisms of action for P in ovulation and in what phase of the ovulatory process P is critical, however, need to be clarified. The present study used a selective P-receptor antagonist, Org 31710, in the in vitro perfused rat ovary model to examine the local role of P and possible effects on prostaglandin (PG) and plasminogen-activator (PA) release in ovulation. Ovaries from eCG (15 IU)-primed rats were perfused for 20 h with LH (0.2 μg/ml) and 3-isobutyl-1-methylxanthine (IBMX, 200 μM) to induce ovulation (median = 10.0, 25%–75% range = 8.5–13). Org 31710 was added at either 0, 3.5, 7, or 9 h after LH IBMX, resulting in significant suppression of ovulation after addition at 0 and 3.5 h (1.0, 1–5.5; and 5.0, 2.5–7.75 ovulations, respectively) but no suppressive effect when added at later time points. Progesterone and estradiol levels in the perfusion media were increased after LH IBMX but were not affected by the presence of Org 31710. Ovarian tissue levels of PGE₂, PGF_2α, and PA activity were measured in ovaries that had been perfused for 10 h, a time that was 2 to 5 h before anticipated ovulation. The presence of Org 31710 significantly decreased the levels of PGE₂, PGF_2α, and PA activity. These results suggest that P is essential in ovulation during the initial stages of the ovulatory process. The effect of P to facilitate ovulation seems to relate to stimulation of the PG- and PA-mediator systems.