The p21-activated kinases (PAKs) play an important role in diverse cellular processes. PAK2 is activated by autophosphorylation upon binding of small G proteins such as Cdc42 and Rac in the GTP-bound state. However, the mechanism of PAK2 autophosphorylation in vitro is unclear. In the present study, the kinetic theory of the substrate reaction during modification of enzyme activity has been applied to a study of the autoactivation of PAK2. On the basis of the kinetic equation of the substrate reaction during the autophosphorylation of PAK2, the activation rate constants for the free enzyme and enzyme-substrate complex have been determined. The results indicate that 1) in the presence of Cdc42, PAK2 autophosphorylation is a bipartite mechanism, with the regulatory domain autophosphorylated at multiple residues, whereas activation coincides with autophosphorylation of the catalytic domain at Thr-402; 2) the autophosphorylation reactions in regulatory domain are either a nonlimiting step or not required for activation of enzyme; 3) the autophosphorylation at site Thr-402 on the catalytic domain occurs by an intermolecular mechanism and is required for phosphorylation of exogenous substrates examined; 4) binding of the exogenous protein/peptide substrates at the active site of PAK2 has little or no effect on the autoactivation of PAK2, suggesting that multiple regions of PAK2 are involved in the enzyme-substrate recognition. The present method also provides a novel approach for studying autophosphorylation reactions. Since the experimental conditions used resemble more closely the in vivo situation where the substrate is constantly being turned over while the enzyme is being modified, this new method would be particularly useful when the regulatory mechanisms of the reversible phosphorylation reaction toward certain enzymes are being assessed.