Fibroblasts transformed by v-src or by related oncogenes encoding activated tyrosine kinases contain elevated levels of polyphosphoinositides with phosphate at the D-3 position of the inositol ring, as a result of the activation of phosphatidylinositol (PI) 3'-kinase. v-sic-transformed cells also contain increased levels of PI 3'-kinase activity immunoprecipitable with anti-phosphotyrosine antibodies; furthermore, PI 3'-kinase can be detected in association with the v-Src tyrosine kinase. To identify regions of v-Src that can interact with PI 3'-kinase, the v-Src SH2 and SH3 domains were expressed in bacteria and incubated with lysates of normal chicken embryo fibroblasts. In vitro, the v-Src SH3 domain, but not the SH2 domain, bound PI 3'-kinase in lysates of uninfected chicken embryo fibroblasts. Substitutions of two highly conserved SH3 residues implicated in ligand binding abolished the ability of the v-Src SH3 domain to associate with PI 3'-kinase. Furthermore, the v-Src SH3 domain bound in vitro to the amino-terminal region of the p85a subunit of PI 3'-kinase. These results suggest that the v-Src SH3 domain may mediate an interaction between the v-Src tyrosine kinase and PI 3'-kinase, by direct binding to p85.Phosphatidylinositol (PI) 3'-kinase was first described in cells transformed by polyomavirus middle-T antigen by virtue of its association with the transforming middle-T-cSrc complex (51). The enzyme has since been detected in association with receptor tyrosine kinases, to which it binds in a ligand-dependent manner (20,22,37,39,41,48 (14,47).PI 3'-kinase consists of a heterodimer of an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110) (4, 31). Sequencing of cDNAs encoding two distinct p85 isoforms (11,35,44) revealed the presence of an SH3 domain at the extreme N terminus and two SH2 domains, one in the middle of the molecule and the other at the extreme C terminus (11,35,44). A region of homology to the Rho/Rac GAP domain of BCR (breakpoint cluster region) is located between the SH3 domain and the N-terminal SH2 domain of p85. The p85 SH2 domains have been implicated in the interaction of PI 3'-kinase with the middle-T antigenc-Src complex (52), with insulin receptor substrate 1 (2), and with members of the platelet-derived growth factor receptor subfamily of tyrosine kinases (19,25,30,38) through binding to the conserved, tyrosine-phosphorylated motif [pY(M/ V)XMJ found in these molecules (3,21,23,38,45,51).