We investigated the effect of insulin on phosphatidylinositol (PtdIns) 3-kinase (PtdIns 3-kinase) activity in neuronal cultures to determine if this enzyme is involved with the neurotrophic actions of insulin. Insulin caused a concentration-dependent increase in PtdIns 3-kinase activity in anti-phosphotyrosine immunoprecipitates. The kinase activity was able to phosphorylate PtdIns, PtdIns 4-phosphate, and PtdIns 4,5-bisphosphate. In intact neurons, a 10-min 1 mM insulin treatment in the presence of [32P]orthophosphate increased the levels of both 3-[32P]PtdIns phosphate and 3,4-[32P]PtdIns bisphosphate by 55 and 193%, respectively. This increase was associated with an increase in neurite outgrowth mediated by insulin. Our results indicate that insulin treatment of neuronal cells in primary culture increases PtdIns 3-kinase activity and the formation of the unique D-3-phosphorylated phosphoinositides, suggesting that growth factor-mediated neuronal growth may include the formation of novel phosphoinositide 3-phosphate phospholipids.