The effect of prostaglandin F2α (PGF) treatment during development of the corpus luteum on concentrations of plasma progesterone, LH, and FSH and the resulting temporal interrelationships among hormones were studied in 72 mares in two experiments. In experiment 1, a single treatment on Day 0 (day of ovulation) or Day 1 was associated with a significant increase in progesterone, but the increase was less (P < 0.05) than in nontreated mares. Eight of 12 mares treated on Day 2 or 3 (combined data) had a progesterone decrease for 1 or 2 days, followed by a gradual resurgence to concentrations similar to those in controls by Day 12. Luteolysis (progesterone decrease to < 1 ng/ml) occurred in each of 18 mares treated on Day 4, 5, or 6, except for one mare with resurgence after treatment on Day 4. In experiment 2, daily PGF treatment on Days 0, 1, and 2 suppressed the progesterone concentrations to < 2 ng/ml through Day 4, followed by an increase, indicating that the luteal cells remained viable during progesterone suppression. When PGF was given on Day 0, the increase in concentration of systemic LH on Day 1 was greater (P < 0.01) than the increase in controls, but the FSH concentration increase was not different from controls. Neither gonadotropin increased significantly after treatment on Day 1 or 2. Both gonadotropins increased within 24 h after PGF treatment on Day 3, 4, 5, or 6. Results indicated that PGF treatment on Day 0 or 1 had a novel retarding effect on progesterone output, treatment on Day 2 or 3 had a transient regressive effect with resurgence to control levels in most mares, and treatment on Day 4, 5, or 6 had a luteolytic effect in almost all mares. The gonadotropin results on Days 0 to 6 are compatible with reported days of change in pituitary content, indicating a direct effect of PGF at the hypothalamo-pituitary area.