How a neuron becomes polarized remains an outstanding question. Here, we report that selection of the future axon among neurites of a cultured hippocampal neuron requires the activity of growth factor receptor tyrosine kinase, phosphatidylinositol 3-kinase (PI 3-kinase), as well as atypical protein kinase C (aPKC). The PI 3-kinase activity, highly localized to the tip of the newly specified axon of stage 3 neurons, is essential for the proper subcellular localization of mPar3, the mammalian homolog of C. elegans polarity protein Par3. Polarized distribution of not only mPar3 but also mPar6 is important for axon formation; ectopic expression of mPar6 or mPar3, or just the N terminus of mPar3, leaves neurons with no axon specified. Thus, neuronal polarity is likely to be controlled by the mPar3/mPar6/aPKC complex and the PI 3-kinase signaling pathway, both serving evolutionarily conserved roles in specifying cell polarity.