The ability of bradykinin to stimulate phosphodiesteratic cleavage of phosphatidylcholine (PC) was investigated in bovine pulmonary artery endothelial cells prelabeled with [3H]choline and [3H]myristic acid. Both labels were preferentially (approximately 80%) incorporated into PC. Bradykinin stimulated a rapid and parallel increase in approximately equivalent amounts of water soluble ([3H]choline plus [3H]phosphocholine) and lipid ([3H]phosphatidic acid plus [3H]diacylglycerol) phosphodiesteratic cleavage products of PC. Formation of the phosphodiesteratic cleavage products occurred prior to the maximum rate of release of prostacyclin into the medium, and ED50 values for both responses were similar (less than 1 nM) and consistent with effects mediated by a high affinity bradykinin receptor. These findings suggest that phosphodiesteratic cleavage of PC may be an important event in the process of receptor-dependent endothelial cell activation.