We have established a simple procedure for the separation of intracellular pool(s) of glucose transporter isoform GLUT-4-containing vesicles from the surface sarcolemma and T tubule membranes of rat skeletal myocytes. This procedure enabled us to immunopurify intracellular GLUT-4-containing vesicles and to demonstrate that 20-30% of the receptors for insulin-like growth factor II/mannose 6-phosphate and transferrin are colocalized with GLUT-4 in the same vesicles. Using our new fractionation procedure as well as cell surface biotinylation, we have shown that these receptors are translocated from their intracellular compartment(s) to the cell surface along with GLUT-4 after insulin stimulation in vivo. Denervation causes a considerable downregulation of GLUT-4 protein in skeletal muscle but does not affect the level of expression of other known component proteins of the corresponding vesicles. Moreover, the sedimentation coefficient of these vesicles remains unchanged by denervation. We suggest that the normal level of GLUT-4 expression is not necessary for the structural organization and insulin-sensitive translocation of its cognate intracellular compartment.