We have investigated the mechanism by which cyclic AMP inhibits PGI2 synthesis in cultured bovine aortic endothelial cells. Inhibition of cyclic AMP phosphodiesterase activity by 3-isobutyl-1-methylxanthine (IBMX) blocks calcium ionophore-induced PGI2 production by 62%. The addition of 3 mM dibutyryl cyclic AMP, alone with IBMX, increases the inhibition to 96%. Release o 3H-arachidonate from membrane phospholipids was inhibited 25% by dibutyryl cyclic AMP, 48% by IBMX, and 76% by isoproterenol plus IBMX. Inhibition by isoproterenol was reversed by 10 micro M propranolol. Release of 3H-arachidonate was also reduced 75% by a combination of 10 micro M PGI2 and 3 mM IBMX. We conclude that hormones like isoproterenol and PGI2 may regulate endothelial cell PGI2 biosynthesis by increasing intracellular cyclic AMP, which then inhibits release of endogenous arachidonate from membrane phospholipids.