We have used two approaches to identify possible substrates of the insulin receptor (IR) tyrosine kinase. First, we used a potent tyrosine phosphatase inhibitor, phenylarsine oxide (PAO), which is reported to be specific for the insulin-induced signal transduction route, to augment tyrosine phosphorylation. Second, we used src homology 2 (SH2) domains fused to glutathione S-transferase as high affinity binding agents for tyrosine-phosphorylated proteins. Using the SH2 domain-containing region of p120 GTPase-activating protein and growth factor-bound protein 2, we observed a tyrosine-phosphorylated M(r) 70,000 protein in insulin- plus PAO-treated NIH3T3 cells overexpressing the IR. This M(r) 70,000 protein, which migrated as a doublet on SDS-polyacrylamide gels, efficiently bound to polyuridylic acid-Sepharose but is distinct from similar-size RNA-binding proteins such as p62 (sam68) and heterogeneous nuclear ribonucleoproteins I, K, L, and M. In addition, it differs from other M(r) 70,000 tyrosine-phosphorylated proteins, such as SH2-containing tyrosine phosphatase, raf1, and paxillin. Tyrosine phosphorylation of this protein was hardly observed after epidermal growth factor treatment. This suggests that the M(r) 70,000 protein is a novel and specific substrate for the IR kinase or an insulin-induced tyrosine kinase. The requirement for PAO to identify this tyrosine phosphorylation indicates a high turnover rate of the tyrosine phosphate.