To investigate the cellular and subcellular distributioi of glucose transporters in skeletal muscle, the gluco transporter isoform GLUT4 was localized in human muscle by electron microscopy via immunogold labeling with monoclonal (1F8) or COOH-terminal peptide polyclonal (ECU4) antibody and in isolated rat membranes by Western blot. There was no labeling c GLUT4 in endothelial cells of the capillaries. There also was no labeling of GLUT4 on the surface plasms membrane (sarcolemma) under either basal or insulin-stimulated conditions. Specific labeling for GLUT4 was clearly observed in two compartments: within the trie (on terminal cisternae and transverse tubules) and on an intracellular compartment, possibly sarcoplasmic tubules. Isolated triad membranes from rat muscle al contained substantial quantities of GLUT4 transporte but there was no detectable GLUT4 protein in isolate sarcolemmal membranes. These data suggest a possible mechanism that involves glucose transport across the muscle cell at the transverse tubule membrane, not the sarcolemma.