To obtain insight into the molecular dynamics and involvement of microtubules and the related signal molecules in the regulation of cell locomotion, we studied the influence of microtubule disruption on actin stress fibers and focal adhesion assembly in addition to cell morphology. We found that all microtubule-disrupting drugs including colcemid and vinblastine rapidly and reversibly induce the formation of actin stress fibers and focal adhesions containing vinculin, accompanied by activated cell motility in serum-starved Balb/c 3T3 cells. In contrast, taxol, a microtubule-stabilizing drug, completely inhibited these effects of the microtubule-disrupting drugs. A microinjection of C3 ADP-ribosyltransferase, a specific inhibitor of rho GTPase, blocked the stress fiber and focal adhesion assembly induced by the microtubule disruption. These results suggested that microtubules contain signal molecules that regulate the formation of stress fibers and focal adhesions by activating the rho signal cascade. We postulate that microtubule-releasing and stress fiber-inducing factors link the intrinsically variable and irregular actin filament dynamics to coordinated and directional locomotion in the process of cell movement.