A member of the p21-activated protein kinase (PAK) family, gamma-PAK has cytostatic properties and is activated by cellular stresses such as hyperosmolarity or DNA damage. We report herein that gamma-PAK is associated in vivo with the nonreceptor protein tyrosine kinase c-Abl. gamma-PAK phosphorylates c-Abl on sites located in the kinase domain, in a region that is implicated in protein-protein interactions and in subcellular localization. Activation of gamma-PAK in human embryonic kidney 293T cells by cotransfection with constitutively active Cdc42 induces activation of c-Abl, resulting in increased phosphotyrosine levels. Cotransfection of c-Abl and gamma-PAK elicits phosphorylation of gamma-PAK on tyrosine and down-regulation of gamma-PAK activity, promoting accumulation of inactive gamma-PAK. gamma-PAK is also phosphorylated in vitro by c-Abl. gamma-PAK activity is regulated by ubiquitination and proteolysis in vivo, as shown by immunoblotting with an anti-ubiquitin antibody in the presence of proteasome inhibitors. In summary, we describe a functional interaction between gamma-PAK and c-Abl in which gamma-PAK stimulates c-Abl tyrosine kinase activity and c-Abl phosphorylates and down-regulates gamma-PAK, suggesting the existence of a negative feedback loop between c-Abl and gamma-PAK.