Activation of the M3 muscarinic receptor in 1321N1 human astrocytoma cells leads to increased phospholipase D (PLD)-catalyzed hydrolysis of phosphatidylcholine, which is maximal within 1 min of exposure to agonist. Studies examining the kinetics of phosphatidylethanol formation indicate that there is no further PLD activation beyond this time. Thrombin, a mitogen for quiescent 1321N1 cells, also activates PLD only transiently. The PLD response does not recover for up to 1 hr and cells that have been exposed to carbachol or thrombin do not respond to subsequent challenge with the heterologous agonist. In contrast to the desensitization observed with agonists, phorbol-12-myristate-13-acetate induces a sustained stimulation of PLD. In addition, cells pretreated with carbachol or thrombin show a normal response to phorbol-12-myristate-13-acetate, suggesting that the enzymatic activity of PLD is not compromised. Guanosine-5'-O-(3-thio)triphosphate activation of PLD in cell-free homogenates is also unaffected by prior treatment of the cells with agonist. Agonist-stimulated PLD activation in 1321N1 cells is mediated by protein kinase C (PKC). Thrombin and carbachol cause comparable changes in redistribution of both PKC-alpha and PKC-epsilon. The increase in membrane-associated PKC-alpha is transient and is reinitiated by addition of the heterologous agonist, whereas PKC-epsilon remains membrane associated for at least 60 min and is not further increased by addition of the heterologous agonist. We suggest that desensitization of PLD activation results from the down-regulation of an as yet undefined mediator required for agonist receptor coupling to PLD and that PKC-epsilon might participate in this down-regulation.