Prostaglandin F_2α (PGF_2α) typically initiates a cascade of events that leads to the functional and structural demise of the corpus luteum. A sheep model was used in which a 1-h, systemic infusion of PGF_2α (20 μg/min) is given at midcycle. Such an infusion mimics the onset of spontaneous luteolysis by causing a transient decrease in peripheral plasma progesterone, which reaches a nadir (∼60% of controls) at 8 h but returns to control levels by 16–24 h. We investigated whether PGF_2α also influenced the endogenous protein levels of tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, and matrix metalloproteinases, MMP-2 and MMP-9, all of which have been implicated in remodeling of the extracellular matrix (ECM). Corpora lutea (Day 11) were collected at 0 h and at 1, 8, 16, and 24 h post-PGF_2α infusion (n = 3 sheep at each time). Immunoblot analysis revealed an immediate and precipitous decline in TIMP-1 (30 kDa) and TIMP-2 (19 kDa) protein levels (60% and 90%, respectively; P < 0.05) at the 1-h time point and remained depressed at 8 h (P < 0.05). Gelatin zymography and other procedures identified three MMPs (85, 70, and 64 kDa), which were shown to be the latent form of MMP-9 and the active and latent forms of MMP-2, respectively. In contrast to the rapid decrease in TIMP-1 and -2 levels, an increase in MMP-2 activity (165% of controls, P < 0.05) occurred at 8 h, which corresponded to the nadir in plasma progesterone. These early changes in TIMPs and MMPs indicate that alterations in the structure of the ECM by PGF_2α may play a hitherto unsuspected role in the subsequent process of functional luteolysis.