The mechanism for prostaglandin (PG) F2 alpha release from pig endometrium after oxytocin (OT) treatment is unknown. OT may rapidly stimulate inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG) formation, consistent with the concept of rapid activation of a second-messenger system. In support of this hypothesis, endometrial IP3 levels were increased (P < 0.05) within 0.5 min after treatment with 0.1 microM OT. In contrast, increased DAG formation was not detected after treatment with OT. However, similar to the stimulation of endometrial PGF2 alpha secretion observed after OT treatment (P < 0.001), PGF2 alpha release was increased (P < 0.01) after treatment with phorbol-12-myristate-13-acetate (PMA), which mimics DAG activation of protein kinase C. Further, stimulation of endometrial PGF2 alpha secretion did not result from cell death induced by PMA or OT because lactate dehydrogenase, a cytosolic marker of cellular integrity, did not leak into the medium after PMA or OT treatment. In contrast, 0.5% saponin (positive control for cell death and concomitant release of lactate dehydrogenase) increased PGF2 alpha secretion (P < 0.05) and lactate dehydrogenase release (P < 0.001). These results indicate that OT induces endometrial IP3 production in a rapid manner indicative of a second-messenger system. The finding that increased DAG was not also detected after OT treatment may reflect rapid metabolism or compartmentalized production of DAG involved in the second-messenger stimulation of phospholipase C. The high background of DAG used in the biosynthesis of cellular lipids would obscure the rather small spatially localized changes in DAG levels resulting from the activation of phospholipase C. The finding that DAG was present at approximately 10 to 20-fold higher levels than IP3 in resting cells was consistent with this conclusion.