Rho small GTPase regulates cell morphology, adhesion and cytokinesis through the actin cytoskeleton, We have identified a protein, pl40mDia, as a downstream effector of Rho, It is a mammalian homolog of Drosophila diaphanous, a protein required for cytokinesis, and belongs to a family of formin-related proteins containing repetitive polyproline stretches. pl40mDia binds selectively to the GTP-bound farm of Rho and also binds to profilin, pl40mDia, profilin and RhoA are co-localized in the spreading lamellae of cultured fibroblasts. They are also co-localized in membrane ruffles of phorbol ester-stimulated sMDCK2 cells, which extend these structures in a Rho-dependent manner, The three proteins are recruited around phagocytic cups induced by fibronectin-coated beads. Their recruitment is not induced after Rho is inactivated by microinjection of botulinum C3 exoenzyme, Overexpression of pl40mDia in COS-7 cells induced homogeneous actin filament formation. These results suggest that Rho regulates actin polymerization by targeting profilin via pl40mDia beneath the specific plasma membranes.