Akt substrate of 160kDa (AS160) is an intermediate in the insulin signalling pathway involved in the regulation of glucose transporter 4 (GLUT4) exocytosis. AS160 is phoshorylated by Akt upon insulin stimulation to inhibit the Rab-GTPase activating protein activity of AS160, resulting in GLUT4 storage vesicle exocytosis. Defects in AS160 signalling and GLUT4 translocation could contribute to the development of insulin resistance. Subcellular fractionation experiments have generated a great deal of data about GLUT4 localisation in healthy and insulin resistant muscle cells, however GLUT4 has never been successfully visualised in human skeletal muscle. These experiments aim to visualise, for the first time, the immunolocalisation of AS160 and GLUT4 in human muscle in fasted conditions, in order to generate further detail about the membrane compartments involved and the cytoplasmic distribution of GLUT4. It is important to first understand the relationship between AS160 and GLUT4 localisation in healthy individuals, to then enable determination of whether there are any AS160/GLUT4 localisation defects in insulin resistant individuals. The immunolocalisation of AS160 and GLUT4 in fasted conditions was investigated in skeletal muscle biopsies (obtained under lidocaine anaesthesia) from 3 lean human males. AS160 and GLUT4 were stained with appropriate antibodies and images were viewed using widefield and confocal immunofluorescence microscopy. Transverse (T) tubules were stained using an antibody against the T tubule-specific calcium channel DHPR, fibre type was determined using an antibody against myosin heavy chain type 1, and an antibody against collagen IV was used to stain the cell basement membrane. GLUT4 staining appeared as bright spots located throughout the cell interior and in the perinuclear and subsarcolemmal regions of human skeletal muscle fibres in fasted conditions. GLUT4 was also localised to regions associated with the T tubules. AS160 generates a positive signal in all muscle fibres and when sections are viewed longitudinally AS160 is also seen localised to regions associated with the T tubules. The images show for the first time the subcellular localisation of GLUT4 and AS160 in human skeletal muscle cells in fasted conditions. They support the hypothesis derived from mouse and rat models that, upon insulin stimulation GLUT4 translocates from intracellular stores in the perinuclear and subsarcolemmal regions, to increase GLUT4 at the plasma membrane and T tubule membranes, allowing insulin-stimulated glucose uptake to occur. This technique will be used to visualise GLUT4 in human muscle in fed conditions to determine and quantify the redistribution of GLUT4, and potentially AS160, that occurs in the insulin-stimulated state. The technique will then be used to establish any defects in AS160 and GLUT4 localisation in fasted and fed conditions in insulin resistant individuals.