Several signaling pathways are activated by interferon alpha (IFN alpha) in hematopoietic cells, including the Jak-Stat and the insulin receptor substrate (TRS) pathways. It has been previously shown that IFN alpha activates the phosphatidylinositol (PI) 3'-kinase via an interaction of the p85 subunit of PI 3'-kinase with IRS proteins. Other studies have proposed that Stat-3 also functions as an adapter for p85. me sought to identify the major pathway that regulates IFN alpha activation of the PI3'-kinase in hematopoietic cells. Our data demonstrate that IFN alpha induces the interaction of p85 with IRS-1 or IRS-2, but not Stat-3, in various hematopoietic cell lines in which IRS-I and/or IRS-S and Stat-3 are activated by IFN alpha. In addition, inhibition of PI 3'-kinase activity by preincubation of cells with the PI S'-kinase inhibitor LY294002 does not affect IFN-dependent formation of SIF complexes that contain Stat-3. To determine whether phosphorylation of tyrosine residues in the IFN receptor is required for activation of the PI 3'-kinase, we performed studies using mouse L929 fibroblasts transfected with mutated human IFNAR1 and/or IFNAR2 subunits of the Type I IFN receptor, lacking tyrosine phosphorylation sites. The serine kinase activity of the PI-3K was activated by human IFN alpha in these cells, suggesting that phosphorylation of the Type I IFN receptor is not essential for PI3K activation. We then determined whether IFN alpha activates the Akt kinase, a known downstream target for PI3'-kinase that mediates anti-apoptotic signals. Akt was activated by insulin or IGF-1, but not IFN alpha, in the IFN alpha-sensitive U-266 myeloma cell line. Altogether, our data establish that the IRS pathway and not the Stat pathway, is the major pathway regulating engagement of PI 3'-kinase in hematopoietic cells. Furthermore, the selective activation of Akt by insulin/IGF-1 suggests the existence of distinct regulatory activities of PI3'-kinase in growth factor versus interferon signaling.