The Ras-related Rho subfamily of GTP-binding proteins (p21s), which includes Rho, Rac and Cdc42Hs, is implicated in different aspects of cytoskeletal organization. These proteins resemble Ras p21 in that their active GTP-bound form is inactivated by intrinsic hydrolysis of the GTP to GDP, which can be stimulated by GTPase-activating proteins (GAPs). ACK, a tyrosine kinase that specifically binds Cdc42Hs in its GTP-bound form, has been described. This binding is mediated by a unique sequence of 47 amino acids C-terminal to an SH3 domain and inhibits both the intrinsic and GAP-stimulated GTPase activity of Cdc42Hs. These findings suggest that ACK may represent a new class of proteins that sustains the GTP-bound active form of the Rho subfamily of GTP binding proteins and which is directly linked to a tyrosine phosphorylation pathway.