The insulin receptor substrate-1 (IRS-1) is rapidly phosphorylated on several tyrosine residues by the activated insulin receptor. Phosphorylated IRS-1 acts as a docking protein for Src homology-2 (SH2) domain-containing proteins involved in insulin signaling. These include in vivo the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3-K) and the phosphotyrosine phosphatase-2C (PTP2C). In this report, we examined which tyrosine residues of IRS-1 are required for the interactions of IRS-1 with PI3-K and PTP2C. To address this issue, we constructed different rat IRS-1 mutants containing mutations in the tyrosine residues that interact with the SH2 domains of PI3-K and PTP2C in vitro. Each of the IRS-1 mutants obtained have been transiently expressed in 293 EBNA cells to study their ability to interact with PI3-K and PTP2C in vivo. Our results demonstrate that mutation of tyrosine 608 affects the PI3-K activity associated with IRS-1, suggesting that this tyrosine is likely to be a principal site of interaction with the SH2 domains of p85 in response to insulin. Furthermore, we found that mutation of tyrosines 1172 and 1222 totally prevents the insulin-induced association of IRS-1 with the SH2 domains of PTP2C, demonstrating that both tyrosines 1172 and 1222 are key elements in the binding sites for the SH2 domains of PTP2C. Finally, we found that the ability of purified PTP2C to dephosphorylate IRS-1 is dependent on the association of PTP2C with phosphorylated IRS-1.