Insulin receptor substrate (IRS) signaling is regulated through serine/threonine phosphorylation, with subsequent IRS degradation. This study examines the differences in IRS-1 and IRS-2 degradation in human neuroblastoma cells. SH-EP cells are glial-like, express low levels of the type I IGF-I receptor (IGF-IR) and IRS-2 and high levels of IRS-1. SH-SY5Y cells are neuroblast-like, with high levels of IGF-IR and IRS-2 but virtually no IRS-1. When stimulated with IGF-I, IRS-1 expression remains constant in SH-EP cells; however, IRS-2 in SH-SY5Y cells shows time- and concentration-dependent degradation, which requires IGF-IR activation. SH-EP cells transfected with IRS-2 and SH-SY5Y cells transfected with IRS-1 show that only IRS-2 is degraded by IGF-I treatment. When SH-EP cells are transfected with IGF-IR or SOCS, IRS-1 is degraded by IGF-I treatment. IRS-1 and -2 degradation are almost completely blocked by PI 3-K inhibitors and partially by proteasome inhibitors. In summary, 1) IRS-2 is more sensitive to IGF-I-mediated degradation, 2) IRS degradation is mediated by PI 3-K and proteasome sensitive pathways, and 3) high levels of IGF-IR, and possibly the subsequent increase in Akt phosphorylation, are required for efficient IRS degradation.