Affinity-purified bovine brain phosphatidylinositol 3-kinase (PI3-kinase) contains two major proteins of 85 and 110 kd. Amino acid sequence analysis and cDNA cloning reveals two related 85 kd proteins (p85α and p85β), which both contain one SH3 and two SH2 regions (src homology regions). When expressed, these 85 kd proteins bind to and are substrates for tyrosine-phosphorylated receptor kinases and the polyoma virus middle-T antigen/pp60^c-src complex, but lack PI3-kinase activity. However, an antiserum raised against p85β immunoprecipitates PI3-kinase activity. The active PI3-kinase complex containing p85α or p85β and the 110 kd protein binds to PDGF but not EGF receptors. p85α and p85β may mediate specific PI3-kinase interactions with a subset of tyrosine kinases.