We have shown previously that insulin stimulated the tyrosine phosphorylation of the alpha-type 85-kDa subunit (p85) of phosphatidylinositol (PI) 3-kinase in vitro and in vivo. In the present work, we identified the major tyrosine phosphorylation sites of the alpha-type p85 by the insulin receptor. [32P]Phosphopeptides obtained from lysylendopeptidase digestion of phosphorylated alpha-type p85 in intact cells after insulin treatment were analyzed using reverse-phase high performance liquid chromatography and thin layer electrophoresis. The tyrosine phosphorylation sites of alpha-type p85 in vivo were assigned to three major phosphopeptides, designated p1, p2, and p3. Highly purified insulin receptor also phosphorylated the purified p85 of PI 3-kinase from the bovine thymus at p1. The purified glutathione S-transferase (GST)-p85 (alpha-type) fusion protein and its truncated proteins from Escherichia coli were also phosphorylated by the purified insulin receptor at p1, p2, and p3 in vitro. Analysis of [32P]phosphopeptide of the truncated GST-p85 (alpha-type) fusion proteins and radiosequence analysis revealed that the p1, p2, and p3 phosphopeptides were phosphorylated at tyrosines 607, 580, and 368, respectively. In addition, phenylalanine substitutions at tyrosine 607 and 580 reduced the p1 and p2 phosphopeptides in vivo, respectively. We conclude that the alpha-type p85 of PI 3-kinase was phosphorylated at tyrosines 368, 580, and 607 by the insulin receptor in vivo.