Tyrosine phosphorylation of cellular proteins is an early and key step after activation of the insulin receptor kinase (IRK). The study of the properties of these proteins should contribute to our understanding of insulin action. In rat hepatoma cells overexpressing human insulin receptors (HTC-IR), insulin treatment resulted in rapid tyrosine phosphorylation of proteins of 180, 94, 68, and 60 kDa. When lysates from insulin-treated cells were immunoprecipitated with anti-Syp antibody, subsequent immunoblotting identified p65 and p68, which reacted with anti-Syp, and p6O and p68, which reacted with antiphosphotyrosine antibody. Thus, insulin treatment yielded tyrosine phosphorylation of both Syp and a Syp-associated p6O molecule. When lysates from insulin-treated cells were adsorbed with a glutathione S-transferase (GST)-Syp-Src homology-2 (SH2) fusion protein, tyrosine- phosphorylated p6O was sequestered. After subjecting lysates to SDS-PAGE, the GST-SypSH2 fusion protein was found to bind to p18O, p94, and p6O. Thus, Syp associates directly with a 60-kDa IRK substrate via its SH2 domains. Syp-associated p6O differed from the 60- to 62-kDa proteins, associating with ras guanosine triphosphatase-activating protein, which also underwent modest tyrosine phosphorylation in response to insulin. Preadsorption of cell lystates with antibody against the 85-kDa subunit (p85) of phosphatidylinositol 3-kinase substantially reduced the amount of p60 subsequently immunoprecipitated by anti-Syp. Thus, p60 associates with both Syp and p85. The amount of tyrosine-phosphorylated p60 exceeded that of p180 in anti-Syp immunoprecipitates, whereas their proportion was comparable in anti-p85 immunoprecipitates. Grb2 was also observed in the anti-Syp immunoprecipitates. When lysates from insulin-treated cells were adsorbed with GST-p85SH2 domains or GST-Grb2, the subsequent eluates contained tyrosine-phosphorylated p60, as determined by immunoblotting with antiphosphotyrosine. Membrane binding assays using GST fusion proteins showed that these associations were direct. Studies in rat liver, muscle, and adipose tissue identified insulin-dependent association of Syp, Grb2, and p85 with tyrosine-phosphorylated p60 in adipose tissue only. We conclude that insulin treatment of HTC-IR cells and rat adipose tissue results in the tyrosine phosphorylation of p60, which might participate in the recruitment of downstream effectors involved in insulin signal transduction.