Prostacyclin (prostaglandin I2) is the major product of arachidonic acid metabolism in vascular cells. Its physiological role may be linked to the ability of the cells to respond continuously with prostaglandin I2 production to a variety of stimuli. We report that human endothelial cells or bovine smooth muscle cells in culture respond with prostaglandin I2 synthesis to a first but not to a second stimulation with arachidonic acid. The development of this refractoriness was independent of the arachidonic acid concentration used (6.6-25 microM) and lasted for about 6 h. The same time was required for the cells to recover completely after inhibition of cyclooxygenase activity by aspirin. Neither cis-polyunsaturated fatty acids (linoleic or oleic acids) nor stearic acid (a long-chain saturated fatty acid) prevented the generation of prostaglandin I2 by arachidonic acid. Similarly to arachidonic acid, thrombin and ionophore A23187 could elicit vascular prostaglandin I2 synthesis only once. Pretreatment of the cells with arachidonic acid rendered the cells unresponsive to any other stimulus. These results indicate that the mechanism of the refractoriness induced by arachidonic acid was different from that induced by the other stimuli. It is proposed that vascular cells cannot be stimulated continuously to produce prostaglandin I2, but this process is regulated by different feedback mechanisms.