Increased matrix metalloproteinase (MMP) expression and activities help to mediate tissue involution through increasing extracellular matrix remodeling and promoting dedifferentiation and, ultimately, apoptosis. Therefore, we hypothesized that prostaglandin (PG) F_2α administration would decrease expression of the tissue inhibitor of metalloproteinase (TIMP)-1, -2, and -3 and effectively increase the MMP:TIMP ratio, leading to glandular involution. In experiment 1, we tested the effects of PGF_2α administration (Day 10 postestrus; Day 0 = estrus) on luteal TIMP-1, -2, and -3 mRNA and protein expression. Corpora lutea were collected at 0, 15, or 30 min or at 1, 2, 4, 6, 12, 24, and 48 h following PGF_2α administration (n = 3–9 animals/time point). Following PGF_2α administration, TIMP-1 mRNA levels decreased (P < 0.05) at 1 and 2 h relative to 0 h (controls), then increased to levels greater than controls at 4 and 6 h. In contrast, TIMP-2 and -3 mRNA levels did not decrease following PGF_2α administration. The TIMP-1, -2, and -3 proteins were localized to large luteal cells (LLCs) within control (untreated) tissues. However, histodepletion of TIMP-1 within LLCs was evident within 30 min (earliest time point collected) following PGF_2α injection and continued through 48 h. Luteal concentration of TIMP-1, as determined by RIA, was decreased (P < 0.05) by 15 min (earliest time point collected) following PGF_2α administration and remained low through 48 h. In contrast, TIMP-2 and -3 immunolocalization was not altered by PGF_2α administration. Experiment 2 was conducted to determine if PGF_2α could initiate the preceding changes in TIMP-1 in early (Day 3) corpora lutea that can bind PGF_2α but are refractory to its luteolytic effects. Serum concentrations of progesterone and luteal concentrations of TIMP-1 mRNA and protein were similar at 0 and 6 h after PGF_2α injection on Day 3 postestrus. These data suggest that an early and sustained effect of PGF_2α is the specific depletion of TIMP-1 within LLCs that are capable of responding to the luteolytic action of PGF_2α. This action may increase the MMP:TIMP-1 ratio, creating an environment that favors extracellular matrix degradation and, thereby, facilitates both functional and structural regression.