Oxytocin (OT) is the physiological stimulus for pulsatile release of endometrial prostaglandin (PG) F2α during luteolysis in domestic ungulates, and the cellular mechanism for this appears to involve phosphoinositide (PI) hydrolysis. To determine which endometrial cell type(s) was responsive to OT during luteolysis in swine, luminal epithelial (LEC), glandular epithelial (GEC), and stromal cells (SC) were isolated from endometrium by differential enzymatic digestion and sieve filtration on Day 16 postestrus and cultured. For PI hydrolysis in experiment 1, SC were most responsive to 100 nM OT (p < 0.001), whereas LEC were least responsive and GEC had an intermediate response (p < 0.001). For PGF secretion in experiment 2, the response to OT was greatest for SC, least for LEC, and intermediate for GEC. In experiment 3, 100 nM OT increased PI hydrolysis in SC within 30 min (p < 0.05) and in GEC within 60 min (p < 0.05) but did not increase PI hydrolysis in LEC. In experiment 4, PI hydrolysis in SC was increased (p < 0.05) by 33–333 nM OT but was not increased by ≤ 333 nM OT in GEC or LEC after 30 min. However, PGF secretion from SC was increased (p < 0.05) by 10–333 nM OT, and from GEC by 10–333 nM OT, but was not increased from LEC by ≤ 333 nM OT. Results of this study indicate that 1) there was differential responsiveness to OT among endometrial cell types, and 2) within cell type, there generally was a similar response to OT for both PI hydrolysis and PGF secretion, further implicating PI hydrolysis as the signaling pathway for OT-stimulated PGF2α release. The differential response of endometrial cell types may have an important role in the pattern of PGF2α secretion during luteolysis in swine.