The present study was undertaken to determine the mechanism by which phorbol ester stimulates eicosanoid synthesis in endothelial cells. We observed that phorbol 12-myristate 13-acetate (PMA) actively stimulated eicosanoid synthesis over a prolonged period of time, and the stimulatory effect was abolished by cycloheximide and actinomycin D. Western blot was employed to test the hypothesis that PMA elicited sustained eicosanoid synthesis via the stimulation of de novo synthesis of prostaglandin G/H synthase (cyclooxygenase, EC 1.14.99.1). Treatment of cultured human umbilical vein endothelial cells resulted in an enhancement of the 70-kDa immunoreactive prostaglandin G/H synthase band over the control cells treated with medium alone. The enhancement was abolished by cycloheximide. Human umbilical vein endothelial cells were then metabolically labeled with L-[35S]methionine, and the effect of PMA on methionine incorporation was evaluated by immunoblotting. PMA increased the synthetic rate of prostaglandin G/H synthase over the control cells. By pulse-chase experiments, we further showed that prostaglandin G/H synthase has a rapid turnover rate (t1/2 less than 10 min) in control cells, and PMA had no effect on the enzyme turnover. Our data indicate that PMA increases the synthesis of prostaglandin G/H synthase which is required for circumventing the autoinactivation of prostaglandin G/H synthase and hence permit sustained conversion of arachidonic acid into eicosanoids.