In order to discover possibly novel insulin receptor substrates and/or downstream targets in the insulin signaling pathway, we established a cell-free system for this purpose using purified insulin receptor and subcellular fractions fi om rat adipocytes as a sourse of cellular substrates. Under these conditions, we have found a 70-kDa protein (pp70) in fat cells that is tyrosine-phosphorylated by the activated insulin receptor. Using sucrose velocity gradient sedimentation we also show that pp70 cofractionate a particulate fraction containing IRS-1 but not with GLUT-4 vesicle-enriched fractions. Our results suggest that pp70 may be an endogenous substrate for the insulin receptor tyrosine kinase.