The activation of p21ras by receptor tyrosine kinases involves the translocation of the growth factor receptor bound protein 2-mammalian son of sevenless protein (Grb2-SOS) complex to the plasma membrane where p21ras is localized. Insulin receptors induce p21ras-GTP formation by two possible mechanisms: tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent association with Grb2, or Shc phosphorylation and its subsequent association with Grb2. We investigated the contribution of the major tyrosine autophosphorylation sites Tyr1158, Tyr1162, and Tyr1163 of the insulin receptor to IRS1.Grb2 and Shc.Grb2 association and the formation of p21ras-GTP. Chinese hamster ovary-derived cell lines were used overexpressing mutant insulin receptors in which the major tyrosine autophosphorylation sites were stepwise replaced by phenylalanines. In cell lines expressing wild type or mutant Y1158F,Y1162,Y1163 (FYY) receptors, insulin stimulated tyrosine phosphorylation of IRS1 and Shc and the formation of IRS1.Grb2 and Shc.Grb2 protein complexes, together with an increase in p21ras-GTP. Cell lines expressing mutant Y1158,Y1162F,Y1163F (YFF) receptors showed insulin-induced tyrosine phosphorylation of Shc, Shc.Grb2 complex formation, and p21ras-GTP formation, whereas tyrosine phosphorylation of IRS1 was strongly decreased and formation of IRS1.Grb2 complexes was undetectable. The activity of FYY and YFF receptors to mediate p21ras-GTP formation correlated with their activity to induce Shc phosphorylation and Shc.Grb2 association. The mutant insulin receptors Y1158F,Y1162F,Y1163 and Y1158F,Y1162F,Y1163F were inactive in inducing any of these responses. We conclude that phosphorylation of Tyr1158 and Tyr1162 of the insulin receptor is linked to distinct post-receptor processes and that YFF receptors generate p21ras-GTP via the Shc.Grb2 pathway rather than one involving IRS1.Grb2 interaction.