IL-9 and IL-4 are cytokines within the IL-2 receptor gamma chain (IL-2Rg) superfamily that possess similar and unique biological functions. The signaling mechanisms, which may determine cytokine specificity and redundancy, are not well understood. IRS proteins are tyrosine phosphorylated following IL-9 and IL-4 stimulation, a process in part mediated by JAK tyrosine kinases (Yin et al. JBC 270:20429, 1995). In the present study, we used 32D cells stably transfected with insulin receptor (32DIR), which do not express any IRS proteins, as a model system to study the requirement of different structural domains of IRS proteins in IL-9 and IL-4 mediated functions. Overexpression of IRS-1 and IRS-2, but not IRS-4, induced proliferation of 32DIR cells in response to IL-9. The PH domain of IRS proteins is required for IRS mediated proliferation stimulated by IL-9. The PTB and SAIN domains are interchangeable for IRS to transduce the proliferative effect of IL-4. Therefore, the PH domain plays different roles in coupling IRS proteins to activated IL-9 and IL-4 receptors. The role of IRS proteins in determining cytokine specificity was corroborated by their ability to interact with different downstream signaling molecules. While PI-3 kinase and Grb-2 interact with tyrosine phosphorylated IRS proteins, Shp-2 only binds to IRS proteins following IL-4, but not IL-9, stimulation. Although PI-3 kinase activity is necessary for the IRS-1/2 mediated proliferative effect of IL-9 and IL-4, Akt activation is only required for cell proliferation induced by IL-4, but not IL-9. These data suggest that IRS-dependent signaling pathways work by recruiting different signaling molecules to determine specificity of IL-2Rg superfamily cytokines.