A diversified series of experiments was conducted to determine the potential role of endothelin-1 (ET-1) in ovine luteal function. Endothelin-1 inhibited basal and LH-stimulated progesterone production by dispersed ovine luteal cells during a 2-h incubation. This inhibition was removed when cells were preincubated with cyclo-d-Asp-Pro-d-Val-Leu-d-Trp (BQ123), a highly specific endothelin ET_A receptor antagonist. Administration of a luteolytic dose of prostaglandin F_2α (PGF_2α) rapidly stimulated gene expression for ET-1 in ovine corpora lutea (CL) collected at midcycle. Intraluteal administration of a single dose of BQ123 to ewes on Day 8 or 9 of the estrous cycle mitigated the luteolytic effect of PGF_2α. Intramuscular administration of 100 μg ET-1 to ewes at midcycle reduced plasma progesterone concentrations for the remainder of the estrous cycle. Following pretreatment with a subluteolytic dose of PGF_2α, i.m. administration of 100 μg ET-1 caused a rapid decline in plasma progesterone and shortened the length of the estrous cycle. These data complement and extend previously published reports in the bovine CL and are the strongest evidence presented to date in support of a role for ET-1 in PGF_2α-mediated luteal function in domestic ruminants.