Grb2-associated binder-1 (Gab1) undergoes tyrosine phosphorylation in response to stimulation by growth factors and hormones including insulin, epidermal growth factor (EGF), nerve growth factor (NGF), and hepatocyte growth factor (HGF). However, the HGF receptor is the only one known to associate directly with Gab1. Herein, we explore the mechanism of Gab1 phosphorylation by other receptor protein-tyrosine kinases unable to bind to Gab1 directly. The Src homology 2 (SH2) domain of the phosphatidylinositol 3-kinase (PI3K) regulatory subunit binds Gab1 in a phosphorylation-independent manner. Moreover, the regulatory subunit of PI3K can mediate the association of Gab1 and receptor protein-tyrosine kinases including the insulin, EGF, and NGF receptors, all of which phosphorylate Gab1. Thus, it appears that the PI3K regulatory subunit acts as an adaptor protein via a phosphotyrosyl-independent SH2 interaction, allowing Gab1 to serve as a substrate for several tyrosine kinases. This is a new role for the PI3K regulatory Subunit.