Both oxytocin (OT) and prostaglandin (PG) possess potent uterotonic activity. It has been suggested that the uterotonic action of OT may be mediated by PG release. We investigated the uterotonic and PG-releasing actions of OT, angiotensin II (AT) and methacholine (MC) in isolated pregnant rat uteri. Our findings indicate that the OT-induced PG release is a direct effect of OT and not a secondary response to myometrial contractions and that the uterotonic action of OT is not dependent on PG participation. This is shown by: 1) equipotent uterotonic doses of OT, AT and MC had different effects on uterine PG release. OT and AT caused uterine contractions and PG release, whereas MC caused contractions but no PG release. 2) OT produced a dose-dependent uterotonic responses. However, there was no proportional relationship and the rate of PG release. 3) In uterine homogenates, which lack the functional integrity for mechanical contractions, OT also caused an increase in PG biosynthesis. Indomethacin suppressed both the spontaneous and the OT-stimulated PG synthesis in the uterine homogenates. 4) In isolated uterine horns, the contractile response to OT was only slightly attenuated in the presence of indomethacin sufficient to inhibit completely the OT-stimulated PG synthesis. We concluded that OT has a dual action in the uterus and may act on two different receptors, one leading to myometrial contractions and the other leading to PG release. AT, an octapeptide like OT, may also have a dual action, whereas the parasympathomimetic, MC, has predominately a direct uterotonic action.