Rho-family GTPases play a central role in the regulation of neuronal morphogenesis. In growth cones, for example, Rho GTPases transduce extracellular stimuli into structural changes such as filopodia and lamellipodia. Although it is generally accepted that Rac1/Cdc42 and RhoA are positive and negative regulators of neurite outgrowth, respectively, the role of each Rho-family member in neuronal morphogenesis may change according to the cell context. At present, the mechanism underlying this complexity is largely unknown. In growth cones, this is partly due to a lack of information on the distribution of active Rho GTPases. Here, we visualized RhoA/Rac1/Cdc42 activities during laminin-induced growth cone advance of DRG neurons and N1E-115 neuroblastoma cells using probes based on fluorescence/Förster resonance energy transfer. The Rac1 and Cdc42 activities were high in the peripheral domain (P-domain) of growth cones. Active Rac1 was uniformly detected throughout the P-domain, whereas Cdc42 activity increased gradually toward the growth cone edge. Against a model involving RhoA down-regulation at the periphery of protruding growth cones, we found that the RhoA activity was higher in the P-domain than in the central domain and axon shaft, and that a high level of RhoA activity was maintained in the extending part of growth cones. In lysophosphatidic acid-treated N1E-115 cells, well-developed neurites with growth cones showed RhoA activation, but sustained their extended morphology until they were drawn toward the contracting somata. On the other hand, suppression of RhoA activity by C3 exoenzyme led to loss or deformation of actin bundles in the growth cones. Thus, RhoA activation in the shaft results in neurite retraction, whereas high RhoA activity in the P-domain is necessary to retain the spread morphology of nerve growth cone.