The effects of insulin stimulation and muscle contractions on the subcellular distribution of GLUT4 in skeletal muscle have been studied on a preparation of single whole fibers from the rat soleus. The fibers were labeled for GLUT4 by a preembedding technique and observed as whole mounts by immunofluorescence microscopy, or after sectioning, by immunogold electron microscopy. The advantage of this preparation for cells of the size of muscle fibers is that it provides global views of the staining from one end of a fiber to the other and from one side to the other through the core of the fiber. In addition, the labeling efficiency is much higher than can be obtained with ultracryosections. In nonstimulated fibers, GLUT4 is excluded from the plasma membrane and T tubules. It is distributed throughout the muscle fibers with ∼23% associated with large structures including multivesicular endosomes located in the TGN region, and 77% with small tubulovesicular structures. The two stimuli cause translocation of GLUT4 to both plasma membrane and T tubules. Quantitation of the immunogold electron microscopy shows that the effects of insulin and contraction are additive and that each stimulus recruits GLUT4 from both large and small depots. Immunofluorescence double labeling for GLUT4 and transferrin receptor (TfR) shows that the small depots can be further subdivided into TfR-positive and TfR-negative elements. Interestingly, we observe that colocalization of TfR and GLUT4 is increased by insulin and decreased by contractions. These results, supported by subcellular fractionation experiments, suggest that TfR-positive depots are only recruited by contractions. We do not find evidence for stimulation-induced unmasking of resident surface membrane GLUT4 transporters or for dilation of the T tubule system (Wang, W., P.A. Hansen, B.A. Marshall, J.O. Holloszy, and M. Mueckler. 1996. J. Cell Biol. 135:415–430).