Neuroblastoma is a heterogeneous tumor consisting of N (neuronal) and S (stromal) cells. We report that more tumorigenic and motile N cells express higher levels of IGF-I receptor (IGF-IR) than less tumorigenic, more adherent S cells. Shc, one of the two major docking partners of IGF-IR, is equally expressed in N and S cell lines. IGF-I treatment phosphorylates Shc in N cells, but only weakly activates Shc in S cells. Expression of the second partner, insulin receptor substrate (IRS), is cell type specific. S cells exclusively express IRS-1 that undergoes sustained phosphorylation by IGF-I. In contrast, N cells express IRS-2 that is transiently phosphorylated by IGF-I. Downstream of IRS-2 and Shc, IGF-I treatment results in strong activation of Akt and MAPK in N cells and activation of both pathways is required for IGF-I-mediated differentiation. Only IGF-IR activation of phosphatidylinositol-3 kinase is required for tumor edge ruffling in N and S cells, with stimulation of focal adhesion kinase (FAK) and paxillin. This detailed understanding of the 'biochemical signature' of N and S cells provides the background needed to target and disrupt specific IGF signaling pathways in an attempt to develop more effective therapies.