The effect of prostaglandin F2α (PGF) infusion for 3 h into the jugular vein on progesterone concentrations was studied in 24 Holstein heifers. Plasma concentrations of PGF were assessed by assay of 13,14-dihydro-15-keto-PGF (PGFM). The 3 h of PGF infusion were used to approximate the duration of the major concentrations of PGFM in a natural pulse. During infusion of 5, 10, or 15 mg/3 h, the concentrations of PGFM greatly exceeded the peak of a natural pulse. Plasma concentrations of progesterone decreased (P < 0.05) in the three PGF-treated groups by Hour 1 (Hour 0 = beginning of infusion). Progesterone increased between Hours 1 and 2, but the increase was significant (P < 0.03) only in the 5-mg group. Concentrations decreased more gradually between Hours 2 and 6 than between Hours 0 and 1 with no differences among PGF groups. The percentage of CL area with color-Doppler signals of blood flow were elevated similarly in the three PGF-treated groups at Hours 1 to 3 and by Hour 5 decreased to below the percentage at Hour 0. In a second experiment, approximating a natural PGFM pulse by intravenous infusion of PGF at a dose of 0.7 mg/3 h did not affect plasma progesterone concentrations. Results indicated that intravenous infusion of PGF for 3 h decreased the progesterone concentration when the total dose was equivalent to doses that have been shown to be completely luteolytic when given as a single systemic injection. However, intravenous infusion of a dose of PGF that approximately simulated a natural PGFM pulse did not effect progesterone concentration.