Signaling through the insulin receptor tyrosine kinase involves its autophosphorylation in response to insulin and the subsequent tyrosine phosphorylation of substrate proteins such as insulin receptor substrate-1 (IRS-1). In basal 3T3-L1 adipocytes, IRS-1 is predominantly membrane-bound, and this localization may be important in targeting downstream signaling elements that mediate insulin action. Since IRS-1 localization to membranes may occur through its association with specific membrane proteins, a 3T3-F442A adipocyte cDNA expression library was screened with non-tyrosine-phosphorylated, baculovirus-expressed IRS-1 in order to identify potential IRS-1 receptors. A cDNA clone that encodes sigma3A, a small subunit of the AP-3 adaptor protein complex, was demonstrated to bind IRS-1 utilizing this cloning strategy. The specific interaction between IRS-1 and sigma3A was further verified by in vitro binding studies employing baculovirus-expressed IRS-1 and a glutathione S-transferase (GST)-sigma3A fusion protein. IRS-1 and sigma3A were found to co-fractionate in a detergent-resistant population of low density membranes isolated from basal 3T3-L1 adipocytes. Importantly, the addition of exogenous purified GST-sigma3A to low density membranes caused the release of virtually all of the IRS-1 bound to these membranes, while GST alone had no effect. These results are consistent with the hypothesis that sigma3A serves as an IRS-1 receptor that may dictate the subcellular localization and the signaling functions of IRS-1.