Lysed aortic smooth muscle cells, when incubated with [14C] arachidonate, synthesized only one radioactive product, which was identified as 6-keto-PGF1alpha. Formation of this product from smooth muscle cell lysates was stimulated when human platelet extracts were added to the system, and further stimulation was observed when imidazole, a selective inhibitor of thromboxane synthesis, was added to this coupled system. These observations indicate that the cyclooxygenase of the smooth muscle cells was rate-limiting, that the prostacyclin synthetase of these cells can utilize endoperoxides produced by platelets, and that blocking of thromboxane synthesis might, under certain conditions, shunt arachidonate metabolism toward prostacyclin formation.