mDial is a downstream target molecule of Rho small G protein and regulates the formation of parallel stress fibers in MDCK cells. mDial consists of at least one Rho-binding domain (RBD), one FH3 domain (FH3D), one coiled-coil domain (CCD), one FH1 domain (FH1D), one FH2 domain (FH2D), and another CCD in this order from the N-terminus to the C-terminus. We constructed various deletion mutants of mDial and investigated the mechanisms of its activation and action by measuring the formation of parallel stress fibers in MDCK cells. We show here that at least FH1D and second CCD are essential for the formation of parallel stress fibers. Furthermore, we present the evidence suggesting that mDial has another domain which interacts with RBD, that this interaction masks FH1D and second CCD and keeps mDial inactive, and that the binding of Rho to RBD opens this folded structure, resulting in the activation of mDial.