Transforming growth factor (TGF)-α- and epidermal growth factor (EGF)-induced signal transduction was directly compared with that of glucose and insulin-like growth factor-1 (IGF-1) in INS-1 cells. TGF-α/EGF transiently (<20 min) induced phosphorylation of extracellular-regulated kinase (Erk)-1/2 (>20-fold), glycogen synthase kinase (GSK)-3 (>10-fold), and protein kinase B (PKB) (Ser⁴⁷³ and Thr³⁰⁸), but did not increase [³H]thymidine incorporation. In contrast, phosphorylation of Erk1/2, GSK-3, and PKB in response to glucose and IGF-1 was more prolonged (>24 h) and, though not as robust as TGF-α/EGF, did increase β-cell proliferation. Phosphorylation of p70^S6K was also increased by IGF-1/glucose, but not by TGF-α/EGF, despite upstream PKB activation. It was found that IGF-1 induced phosphatidylinositol 3-kinase (PI3K) association with insulin receptor substrate (IRS)-1 and -2 in a glucose-dependent manner, whereas TGF-α/EGF did not. The importance of specific IRS-2-mediated signaling events was emphasized in that adenoviral-mediated overexpression of IRS-2 further increased glucose/IGF-1-induced β-cell proliferation (more than twofold; P < 0.05) compared with control or adenoviral-mediated IRS-1 overexpressing INS-1 cells. Neither IRS-1 nor IRS-2 overexpression induced a β-cell proliferative response to TGF-α/EGF. Thus, a prolonged activation of Erk1/2 and PI3K signaling pathways is important in committing a β-cell to a mitogenic event, and it is likely that this sustained activation is instigated by signal transduction occurring specifically through IRS-2.