In order to identify novel substrates involved in insulin receptor signaling, a yeast two-hybrid 3T3-L1 adipocyte cDNA library was screened with the cytoplasmic domain of the human insulin receptor as bait. Here we describe the isolation and characterization of an interacting protein, APS, which contains pleckstrin homology and Src homology 2 domains and several potential tyrosine phosphorylation sites. APS mRNA and protein are expressed primarily in skeletal muscle, heart, and adipose tissue, and in differentiated 3T3-L1 adipocytes. We show that APS associates with phosphotyrosines situated within the activation loop of the insulin receptor via the APS Src homology 2 domain. Insulin stimulation of 3T3-L1 adipocytes resulted in rapid tyrosine phosphorylation of endogenous APS on tyrosine 618, whereas platelet-derived growth factor treatment resulted in no APS phosphorylation. In summary, we have identified a new insulin receptor substrate that is primarily expressed in insulin-responsive tissues and in 3T3-L1 adipocytes whose phosphorylation shows insulin receptor specificity. These findings suggest a potential role for APS in insulin-regulated metabolic signaling pathways.