The p21-activated kinases (PAKs) are important mediators of cytoskeletal reorganization, cell motility and transcriptional events regulated by the Rho family GTPases Rac and Cdc42. PAK activation by serum components is strongly dependent on cell adhesion to the extracellular matrix (ECM). PAK binds directly to the Nck adapter protein, an interaction thought to play an important role in regulation and localization of PAK activity. This report demonstrates that the interaction of PAK with Nck is regulated dynamically by cell adhesion. PAK-Nck binding is rapidly lost after cell detachment and rapidly restored after re-adhesion to the ECM protein fibronectin, suggesting a rapidly reversible mode of regulation. Furthermore, the loss of Nck binding correlates with changes in the phosphorylation state of PAK in nonadherent cells, as evidenced by electrophoretic mobility shift and phosphorylation within a sequence known to mediate interaction with Nck. The ability of cell adhesion to regulate PAK phosphorylation and interaction with Nck may contribute to the anchorage-dependence of PAK activation as well as to the localization of activated PAK within a cell.