Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by association with a variety of tyrosine kinase receptors and intracellular tyrosine-phosphorylated substrates. We isolated a cDNA that encodes a 50-kDa regulatory subunit of PI 3-kinase with an expression cloning method using 32 P-labeled insulin receptor substrate-1 (IRS-1). This 50-kDa protein contains two SH2 domains and an inter-SH2 domain of p85␣, but the SH3 and bcr homology domains of p85␣ were replaced by a unique 6-amino acid sequence. Thus, this protein appears to be generated by alternative splicing of the p85␣ gene product. We suggest that this protein be called p50␣. Northern blotting using a specific DNA probe corresponding to p50␣ revealed 6.0-and 2.8-kb bands in hepatic, brain, and renal tissues. The expression of p50␣ protein and its associated PI 3-kinase were detected in lysates prepared from the liver, brain, and muscle using a specific antibody against p50␣. Taken together, these observations indicate that the p85␣ gene actually generates three protein products of 85, 55, and 50 kDa. The distributions of the three proteins (p85␣, p55␣, and p50␣), in various rat tissues and also in various brain compartments, were found to be different. Interestingly, p50␣ forms a heterodimer with p110 that can as well as cannot be labeled with wortmannin, whereas p85␣ and p55␣ associate only with p110 that can be wortmanninlabeled. Furthermore, p50␣ exhibits a markedly higher capacity for activation of associated PI 3-kinase via insulin stimulation and has a higher affinity for tyrosinephosphorylated IRS-1 than the other isoforms. Considering the high level of p50␣ expression in the liver and its marked responsiveness to insulin, p50␣ appears to play an important role in the activation of hepatic PI 3-kinase. Each of the three ␣ isoforms has a different function and may have specific roles in various tissues.A variety of growth factors and hormones mediate their cellular effects via interactions with cell surface receptors that possess protein kinase activity (1, 2). The interaction of most of these ligands with their receptors induces tyrosine kinase activation and autophosphorylation of the receptor, resulting in physical association of these receptors with several cytoplasmic substrates having SH2 domains. Phosphatidylinositol 3-kinase (PI 3-kinase) 1 has been identified through its ability to associate with cellular protein kinases, including numerous growth factor receptors and oncogene products (3, 4). This lipid kinase phosphorylates phosphatidylinositol at the D-3 position of the inositol ring in response to stimulation with a variety of growth factors and hormones (5). Although the role of this lipid product in cellular regulation remains unclear, recent reports suggest that the activation of PI 3-kinase leads to the activation of c-Akt, Rac, PKC-␥ isoform, and p70 S6 kinase (6 -9). As a result, PI 3-kinase has been suggested to play essential roles in the regulation of various cellular activities, including proliferation (10, 11), differen...