Actin depolymerization through Rho GTPases or exogenous mechanical tension has been suggested as a key determinant for the first step of neuronal polarization, the axonogenesis, in which one of the neurites starts to grow becoming the axon. The underlying mechanism and the relationship between two forces in the cells, however, are mostly unknown. Here, we report that the myosin-dependent contractility is a common effector between two forces and a critical determinant in axonogenesis and neuronal polarization. We have found that inhibition of myosin ATPase activity and modulation of myosin light chain phosphorylation/dephosphorylation through Rho GTPases signaling induced multiple axons. Moreover, overexpression of wild-type myosin light chain kinase dramatically increased filopodial structures and produced multi-axonal Structures. Our results suggest that MLC phosphorylation/dephosphorylation through Rho GTPases signaling modulates the actomyosin contractility, and then in turn provides a physiological tension in neurons to induce axon. (C) 2004 Elsevier Inc. All rights reserved.