This study examines differences in intracellular responses to cloprostenol, a prostaglandin (PG)F_2α analog, in porcine corpora lutea (CL) before (Day 9 of estrous cycle) and after (Day 17 of pseudopregnancy) acquisition of luteolytic capacity. Pigs on Day 9 or Day 17 were treated with saline or 500 μg cloprostenol, and CL were collected 10 h (experiment I) or 0.5 h (experiment III) after treatment. Some CL were cut into small pieces and cultured to measure progesterone and PGF_2α secretion. In experiment I, progesterone remained high and PGF_2α low in luteal incubations from either Day 9 or Day 17 saline-treated pigs. Cloprostenol increased PGF_2α production 465% and decreased progesterone production 87% only from Day 17 luteal tissue. Cloprostenol induced prostaglandin G/H synthase (PGHS)-2 mRNA (0.5 h) and protein (10 h) in both groups. In cell culture, cloprostenol or phorbol 12,13-didecanoate (PDD) (protein kinase C activator), induced PGHS-2 mRNA in luteal cells from both groups. However, acute cloprostenol treatment (10 min) decreased progesterone production and increased PGF_2α production only from Day 17 luteal cells. Thus, PGF_2α production is induced by cloprostenol in porcine CL with luteolytic capacity (Day 17) but not in CL without luteolytic capacity (Day 9). However, this change in PGF_2α production is not explained by a difference in induction of PGHS-2 mRNA or protein.