Responses to intravenous treatment of mares with prostaglandin F2alpha (PGF) 8 d after ovulation were studied in three groups (n=4/group): control (no treatment), bolus (single treatment with 2.5 mg PGF), and infusion (0.1 mg PGF during 2 h). Infusion resulted in a 13,14-dihydro-15-keto-PGF2alpha (PGFM) concentration (559+/-44 pg/mL) that was not different from the mean concentration for the major portion of a natural PGFM pulse associated with luteolysis (569+/-45 pg/mL; n=5). Progesterone in the bolus group increased (P<0.03) between 0 (17.8+/-3.5 ng/mL) and 2 min (25.3+/-4.8 ng/mL), peaked at 10 min (28.5+/-4.6 ng/mL), and then decreased. In the infusion group, progesterone decreased (P<0.05) during 1 min (17.2+/-1.3 ng/mL) to 15 min (13.5+/-1.5 ng/mL) after the beginning of infusion and decreased (P<0.05) similarly to the bolus group during 2 to 12 h; concentrations were lower (P<0.05) at each hour than in controls. Interval between ovulations was shorter (P<0.05) in the bolus (19.3+/-2.0 d) and infusion (18.8+/-2.1 d) groups than in controls (24.3+/-1.3 d). Concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and cortisol increased (P<0.05) within 10 min in the bolus group but did not change in the infusion group. Results supported the hypothesis that increases in progesterone, FSH, LH, and cortisol after a bolus luteolytic PGF treatment are nonphysiologic. Past conclusions on the nature of the luteolytic mechanism are problematic if based on responses to treatment with a single luteolytic bolus of PGF.