Porcine aortic endothelial cells studied at confluence were found to synthesize both prostacyclin and prostaglandin E2. Addition of arachidonic acid, bradykinin, the calcium ionophore A23187 or thrombin stimulated prostaglandin formation, whereas addition of angiotensin II did not. Bradykinin was found to stimulate very potently arachidonic acid release from cells prelabelled with [3H]arachidonate, the response being dose-dependent and half-maximal at 8 ng/ml. The rate of release of label (primarily arachidonate) from cells was increased by bradykinin (100 ng/ml) approximately 8-fold, with a return to control levels by 10 min. The calcium ionophore, A23187, similarly released [3H]arachidonic acid from prelabelled cells; the rate of release was approximately linear for 15 min. Both bradykinin and ionophore A23187 stimulated [3H]arachidonate release from endothelial cell phospholipids, an effect which was abolished in a dose-dependent manner by mepacrine. Release in response to bradykinin was prevented by incubation in Ca2+-free medium. Trifluoperazine, a compound which can inhibit calmodulin-mediated events, blocked the release of label stimulated by bradykinin. These data indicate that the likely mechanism of bradykinin-stimulated prostaglandin production in endothelial cells involves the activation of a phospholipase via a Ca2+-calmodulin-dependent pathway.