Recent experiments in various cell types such as mammalian neutrophils and Dictyostelium discoideum amoebae point to a key role for the lipid product of PI 3-kinase, PIP(3), in determining internal polarity. In neurons, as a consequence of the elongation of one neurite, the axon is specified and the cell acquires its polarity. To test the hypothesis that PI 3-kinase and PIP(3) may play a role in neuronal polarity, and especially in axon specification, we observed localization of PIP(3) visualized by Akt-PH-GFP in developing hippocampal neurons. We found that PIP(3) accumulates in the tip of the growing processes. This accumulation is inhibited by addition of PI 3-kinase inhibitors. Those inhibitors, consistently with a role of PIP(3) in process formation and elongation, delay the transition from stage 1 neurons to stage 3 neurons, and both axon formation and elongation. Moreover, when the immature neurite contacts a bead coated with laminin, a substrate known to induce axon specification, PIP(3) accumulates in its growth cone followed by a rapid elongation of the neurite. In such conditions, the addition of PI 3-kinase inhibitors inhibits both PIP(3) accumulation and future axon elongation. These results suggest that PIP(3) is involved in axon specification, possibly by stimulating neurite outgrowth. In addition, when a second neurite contacted the beads, this neurite rapidly elongates whereas the elongation of the first laminin-contacting neurite stops, consistently with the hypothesis of a negative feedback mechanism from the growing future axon to the other neurites.