: The mechanism whereby agonist occupancy of muscarinic cholinergic receptors elicits an increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin has been examined. Addition of oxotremorine‐M to SH‐SY5Y neuroblastoma cells resulted in rapid increases in the phosphorylation of FAK (t1/2 = 2 min) and paxillin that were independent of integrin‐extracellular matrix interactions, cell attachment, and the production of phosphoinositide‐derived second messengers. In contrast, the increased tyrosine phosphorylations of FAK and paxillin were inhibited by inclusion of either cytochalasin D or mevastatin, agents that disrupt the cytoskeleton. Furthermore, phosphorylation of FAK and paxillin could be prevented by addition of either wortmannin or LY‐294002, under conditions in which the synthesis of phosphatidylinositol 4‐phosphate was markedly attenuated. These results indicate that muscarinic receptor‐mediated increases in the tyrosine phosphorylation of FAK and paxillin in SH‐SY5Y neuroblastoma cells depend on both the maintenance of an actin cytoskeleton and the ability of these cells to synthesize phosphoinositides.