A new in vivo model for studying luteolysis was developed in sheep to provide a convenient method for collecting corpora lutea for molecular, biochemical, and histological analysis during a procedure that mimics natural luteolysis. It was found that the infusion of prostaglandin F(2α) (PGF(2α)) at 20 μg/min/h into the systemic circulation during the mid luteal phase of the cycle allowed sufficient PGF(2α) to escape across the lungs and thus mimic the transient 40% decline in the concentration of progesterone in peripheral plasma seen at the onset of natural luteolysis in sheep. Additional 1h-long systemic infusions of PGF(2α), given at physiological intervals, indicated that two infusions were not sufficient to induce luteolysis. However, an early onset of luteolysis and estrus was induced in one out of three sheep with three infusions, two out of three sheep with four infusions, and three out of three sheep with five infusions. Reducing the duration of each systemic infusion of PGF(2α) from 1h to 30 min failed to induce luteolysis and estrus even after six systemic infusions indicating that, not only are the amplitude and frequency of PGF(2α) pulses essential for luteolysis, but the actual duration of each pulse is also critical. We conclude that a minimum of five systemic pulses of PGF(2α), given in an appropriate amount and at a physiological frequency and duration, are required to mimic luteolysis consistently in all sheep. The five pulse regimen thus provides a new accurate in vivo model for studying molecular mechanisms of luteolysis.