The effects of purinergic agonists on phosphatidylcholine (PC) breakdown and prostacyclin synthesis were investigated in cultured bovine pulmonary artery endothelial cells (BPAEC) prelabeled with [3H]choline and [14C]myristic acid. Both labels were selectively incorporated into PC. In BPAEC prelabeled with [3H]choline, ATP stimulated a rapid 5-fold increase in intracellular free [3H]choline. [3H]Choline formation was associated with a concomitant loss of 3H from PC, and it was not preceded by an increase in the 3H content of other PC degradation products. In BPAEC prelabeled with [14C]myristic acid, ATP stimulated a rapid increase in [14C]phosphatidic acid and [14C]diacylglycerol. These changes were associated with a loss of 14C from PC but not from phosphatidylinositol. In permeabilized BPAEC prelabeled with [3H]choline, ATP and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) stimulated an increase in [3H]choline but not [3H]phosphocholine. The effects of ATP and GTP gamma S were synergistic at low GTP gamma S concentrations. Permeabilized BPAEC did not convert exogenous [3H]phosphocholine to [3H]choline. These data are consistent with the notion that purinergic agonists stimulate PC breakdown by a phospholipase D mechanism. This suggestion was supported by the observation that in the presence of ethanol, ATP stimulated the formation of [14C]phosphatidylethanol in BPAEC prelabeled with [14C]myristic acid. Several adenine nucleotides and related purine derivatives were compared for their effectiveness in stimulating PC breakdown and prostacyclin synthesis. The chemical specificity observed for the two responses indicated that both were mediated by the P2Y purinoceptor subtype. PC breakdown stimulated by ATP occurred prior to the maximum rate of release of prostacyclin into the medium. Taken together these data indicate that PC breakdown in endothelial cells stimulated with purinergic agonists is due, at least in part, to the activation of a phospholipase D that is coupled to purinoceptors by a guanine nucleotide-binding protein. In conjunction with previous data on the effects of bradykinin, these findings suggest an important role for PC breakdown in the mechanism of signal transduction in endothelial cells stimulated with Ca2+-mobilizing agonists.