In a previous study, we have shown that extracellular ATP stimulates Ca 2+ influx resulting in the release of arachidonic acid (AA) and prostaglandin E 2 (PGE 2) synthesis in osteoblast-like MC3T3-E1 cells. In addition, we have recently reported that extracellular ATP stimulates phosphatidylcholine hydrolysis by phospholipase D (PLD) independently from the activation of protein kinase C in these cells. It is well recognized that phosphatidylcholine is hydrolysed by PLD, generating phosphatidic acid, which can be further degraded by phosphatidic acid phosphohydrolase to diacylglycerol (DG). In the present study, we investigated the role of PLD activation in the extracellular ATP-induced AA release and PGE 2 synthesis in osteoblast-like MC3T3-E1 cells. Extracellular ATP stimulated AA release dose-dependently in the range between 0.1 and 1 mM. Propranolol, which is known to inhibit phosphatidic acid phosphohydrolase, significantly inhibited the AA release induced by extracellular ATP in a dose-dependent manner in the range between 100 and 300 μM. 1,6-Bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a selective inhibitor of DG lipase, significantly suppressed the AA release induced by extracellular ATP. Both the pretreatment of propranolol and RHC-80267 also inhibited the extracellular ATP-induced PGE 2 synthesis. These results strongly suggest that the AA release induced by extracellular ATP is mediated at least in part by phosphatidylcholine hydrolysis by PLD in osteoblast-like cells.