We studied the heterologous desensitization of muscarinic receptors by ATP in fura-2-loaded rat parotid acinar cells. Exposure to ATP or 3′-o-(4-benzoyl) benzoyl-ATP shortened the duration and decreased the magnitude of acetylcholine-induced Ca²⁺ release from intracellular Ca²⁺ stores in a dose-dependent manner. The shortening was observed only in an early stage of desensitization (within 20 s), whereas the decrease in the magnitude of the response was dependent upon the time the cells were exposed to the nucleotides. Atropine induced a profound shortening during the progressive decrease in the magnitude of acetylcholine-induced Ca²⁺ release. 3′-o-(4-Benzoyl) benzoyl-ATP did not induce an increase in the cytosolic Ca²⁺ concentration when the cells were incubated in the Ca²⁺- and Na⁺-free medium, but it did induce a strong desensitization of muscarinic receptors. The specific protein kinase C inhibitor bisindoylmaleimide resensitized the 3′-o-(4-benzoyl) benzoyl-ATP-treated muscarinic receptors. Phorbol 12-myristate 13-acetate potentiated the desensitization of muscarinic receptors. Ceramides that prevent the activation of phospholipase D resensitized the 3′-o-(4-benzoyl) benzoyl-ATP-treated muscarinic receptors. These results suggest that ATP, acting through P_2Z purinoceptor-mediated phospholipase D, may produce a Ca²⁺-independent protein kinase C. Heterologous desensitization of muscarinic receptors by protein kinase C may shorten the duration and decrease the magnitude of acetylcholine-induced Ca²⁺ release.