In ruminants, endometrial prostaglandin F₂ (PGF₂) is the luteolytic hormone. Cellular transport of PGF₂ in the uterine endometrium is critical for regulation of the estrous cycle. Molecular mechanisms responsible for control of PGF₂ transport in endometrium during luteolysis are largely unknown. In the present study, we characterized the prostaglandin transporter (PGT) in ovine endometrium. Ovine PGT cDNA consists of 1935 nucleotides that encode 644 amino acids. In ovine endometria, PGT is highly expressed during the period of luteolysis, between Days 14 and 16 of the estrous cycle, in luminal and glandular epithelia. Pharmacological and genomic inhibition of PGT indicates that it is responsible for influx and efflux of PGF₂ in ovine endometrial epithelial cells. Inhibition of PGT during the period of luteolysis prevents the release of oxytocin-induced PGF₂ pulses and maintains functional corpus luteum and its secretion of progesterone. In ovine endometrial epithelial cells, protein kinase A (PKA) and protein kinase C (PKC) pathways are involved in regulating the influx of PGF₂ whereas epidermal growth factor receptor (EGFR) pathways are implicated in regulation of influx and efflux of PGF_2. The extracellular regulated kinases 1/2 (ERK1/2) pathway is associated with efflux of PGF₂ while Jun-amino-terminal kinase /stress-activated protein kinase (JNK/SAPK) pathways are involved in both efflux and influx of PGF_2. Phosphatidylinositol 3-kinase (PI3K) pathways are not involved in either influx or efflux of PGF₂ in ovine endometrial epithelial cells. These are the first results to demonstrate a functional role for PGT in regulation of PGF₂ efflux and influx in ovine endometrial cells that influence luteolytic mechanisms in ruminants.