We have recently demonstrated that c-fes protooncogene product (FES), or a FES-related protein, associates with the interleukin-4 receptor alpha chain (IL-4R alpha) and phosphatidylinositol-3 (PI3) kinase in mouse T cell lines; however, others have demonstrated that PI3 kinase associates with IL-4R alpha through tyrosine phosphorylated insulin receptor substrate (IRS)-2 in other cell types. In order to examine whether IL-4 activates these two distinct PI3 kinase pathways in the same cells, we analyzed association of PI3 kinase with IRS-2, and tyrosine phosphorylation of IRS-2, in a mouse pro-B cell line, Ba/F3, and a mouse mast cell line, MC9. In both cell lines, IL-4 induced tyrosine phosphorylation of IRS-2, association of PI3 kinase with IRS-2, and FES or a FES-related protein. These results indicate that IL-4 activates two distinct PI3 kinase pathways in the same cells. We further identified the critical region in the cytoplasmic domain of IL-4R alpha required for tyrosine phosphorylation of IRS-2.