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 32P-labeled insulin receptor substrate-1 (IRS-1). This 50-kDa protein contains two SH2 domains and an inter-SH2 domain of p85alpha, but the SH3 and bcr homology domains of p85alpha were replaced by a unique 6-amino acid sequence. Thus, this protein appears to be generated by alternative splicing of the p85alpha gene product. We suggest that this protein be called p50alpha. Northern blotting using a specific DNA probe corresponding to p50alpha revealed 6.0- and 2.8-kb bands in hepatic, brain, and renal tissues. The expression of p50alpha protein and its associated PI 3-kinase were detected in lysates prepared from the liver, brain, and muscle using a specific antibody against p50alpha. Taken together, these observations indicate that the p85alpha gene actually generates three protein products of 85, 55, and 50 kDa. The distributions of the three proteins (p85alpha, p55alpha, and p50alpha), in various rat tissues and also in various brain compartments, were found to be different. Interestingly, p50alpha forms a heterodimer with p110 that can as well as cannot be labeled with wortmannin, whereas p85alpha and p55alpha associate only with p110 that can be wortmannin-labeled. Furthermore, p50alpha exhibits a markedly higher capacity for activation of associated PI 3-kinase via insulin stimulation and has a higher affinity for tyrosine-phosphorylated IRS-1 than the other isoforms. Considering the high level of p50alpha expression in the liver and its marked responsiveness to insulin, p50alpha appears to play an important role in the activation of hepatic PI 3-kinase. Each of the three alpha isoforms has a different function and may have specific roles in various tissues.