The establishment of a polarized morphology is an essential event in the differentiation of neurons into a single axon and dendrites. We previously showed that glycogen synthase kinase-3 beta (GSK-3 beta) is critical for specifying axon/dendrite fate by the regulation of the phosphorylation of collapsin response mediator protein-2 (CRMP-2). Here, we found that the overexpression of the small GTPase Ras induced the formation of multiple axons in cultured hippocampal neurons, whereas the ectopic expression of the dominant negative form of Ras inhibited the formation of axons. Inhibition of phosphatidylinositol-3-kinase (PI3-kinase) or extracellular signal-related kinase (ERK) kinase (MEK) suppressed the Ras-induced formation of multiple axons. The expression of the constitutively active form of PI3-kinase or Akt (also called protein kinase B) induced the formation of multiple axons. The overexpression of Ras prevented the phosphorylation of CRMP-2 by GSK-3p. Taken together, these results suggest that Ras plays critical roles in establishing neuronal polarity upstream of the PI3-kinase/Akt/GSK-3 beta/CRMP-2 pathway and mitogen-activated protein kinase cascade. (c) 2005 Elsevier Inc. All rights reserved.