These studies were undertaken to determine how treatment with 100 nM progesterone and/or 10 nM oestradiol-17β acutely (3 h; Experiment 1) or chronically (72 h; Experiments 2–4) influenced basal and oxytocin (OT)-stimulated prostaglandin (PG) F_2α secretion, in enriched cultures of pig endometrial luminal epithelial, glandular epithelial and stromal cells obtained on Day 16 (Experiments 1, 2 and 4) or Day 12 (Experiment 3) after oestrus. In Experiment 1, acute treatment with progesterone stimulated PGF_2α secretion from each cell type on Day 16, whereas acute oestradiol treatment inhibited the stimulatory action of progesterone on PGF_2α secretion only in glandular epithelial cells. In Experiment 2, OT stimulated phospholipase (PL) C activity in luminal epithelial cells on Day 16 only in the presence of chronic oestradiol treatment. For glandular epithelial cells on Day 16, OT stimulated PLC activity only in the presence of chronic treatment with steroid. In stromal cells on Day 16, OT stimulated PLC activity in the absence of steroids and the response to OT was further enhanced by oestradiol. In the absence of chronic treatment with steroid, OT did not stimulate PGF_2α secretion from luminal epithelial cells, but oestradiol induced a response to OT. For glandular epithelial cells, OT-induced PGF_2α secretion was not altered by steroids, whereas the stimulatory response to OT was inhibited by oestradiol or progesterone in stromal cells. For endometrial cells obtained on Day 12 after oestrus in Experiment 3, OT only stimulated PGF_2α release from glandular epithelial and stromal cells. For luminal epithelial cells obtained on Day 16 after oestrus and cultured under polarizing conditions in Experiment 4, secretion of PGF_2α occurred preferentially from the basolateral surface and was stimulated by OT more from the basolateral surface than from the apical surface. Oxytocin-induced PGF_2α secretion from the apical surface was enhanced by chronic treatment with oestradiol, whereas that from the basolateral surface was enhanced by chronic treatment with progesterone. In summary, oestradiol enhanced OT-induced PGF_2α secretion from the apical surface of luminal epithelial cells and reduced the response of stromal cells to OT, actions that may contribute to the reorientation of PGF_2α from endocrine secretion (i.e. towards the uterine vasculature) to exocrine secretion (i.e. towards the uterine lumen) during pregnancy recognition in pigs.