Receptor signalling to phospholipase D (PLD) in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor apparently involves Rho proteins. Since phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been recognized as an essential cofactor for PLD activity and since activated Rho proteins have been reported to stimulate the synthesis of PtdIns(4,5)P2, we studied whether in HEK cells PLD activity is regulated by PtdIns(4,5)P2 and, in particular, whether PtdIns(4,5)P2 can restore PLD activity inhibited by Clostridiurn difficile toxin B, which inactivates Rho Hydrolysis of membrane phospholipids by phospholipases is a common signalling pathway of cell surface receptors. Cleavage of the minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], by phospholipase C isoenzymes, generating the two second messengers, inositol trisphosphate and diacylglycerol, is one of best studied signal transduction pathways [l, 21. Hydrolysis of the major membrane phospholipid, phosphatidylcholine (PtdCho), to phosphatidic acid and choline by phospholipase D (PLD) is another common response to activation of cell surface receptors and is observed in a wide range of cell types [3, 41. In contrast to regulation of phospholipase C, however, the components and mechanisms involved in receptor signalling to PLD are only poorly defined. Besides the receptor themselves, various intracellular compoCorrespondence to