A 60-kDa tyrosine-phosphorylated protein has been observed after insulin treatment of cells in immunoprecipitations of the GTPase-activating protein of Ras (called GAP) as well as the phosphatidylinositol 3-kinase. In the present studies, these two 60-kDa proteins have been shown to differ by limited proteolytic digestions as well as by immunoprecipitation with a monoclonal antibody. This monoclonal antibody was also utilized to show that the 60-kDa GAP-associated protein was rapidly phosphorylated in intact cells after insulin stimulation and to associate with GAP only after insulin treatment of the cells. In addition, the 60-kDa protein was found to be phosphorylated in vitro by the insulin receptor. Finally, the 60-kDa protein immunoprecipitated by this antibody was found not to react with a polyclonal antibody directed against a 62-kDa tyrosine-phosphorylated GAP-associated protein previously observed in src-transformed cells. These studies indicate that insulin stimulates the tyrosine phosphorylation of at least two distinct 60-kDa proteins, one that becomes associated with GAP and appears to be a direct substrate of the insulin receptor kinase and another that associates with the phosphatidylinositol 3-kinase.