In skeletal muscle, both insulin and muscle contractions mediate translocation of glucose transporter GLUT4 to the plasma membrane proper, the sarcolemma, and the specialized membrane channel network, the transverse (t)-tubules. Despite the fact that skeletal muscle glucose uptake plays a major role in normal conditions, in insulin resistance, and type II diabetes, the details of GLUT4 translocation and the intracellular signalling involved have not been fully described. A main reason is that the majority of experiments have been carried out in muscle cultures in vitro. In vitro cultured muscle is not fully differentiated and, therefore, diverges from real muscle, in that it has lower expression of GLUT4, an underdeveloped or nonexistent t-tubule network, and a reduced or nonexistent response to insulin. Thus, experiments carried out in cultured muscle cell systems might give misleading results on how GLUT4 translocation and the signalling involved takes place. To address this problem, a confocal imaging technique has been developed that allows delineation of the spartial and spatial distribution of GFP-tagged GLUT4 (GLUT4-GFP) translocation in living muscle fibers in situ in anesthetized mice. The effects of stimuli with insulin or in situ muscle contractions in fully differentiated muscle fibers can now be studied before, during, and after applying stimuli. Initial analysis of insulin-stimulated GLUT4-GFP translocation showed a delay in maximal translocation between the sarcolemma and t-tubules. Corresponding to the delay, we found that fluorescent tagged insulin reaches the sarcolemma first and then, with a delay, diffuses into the t-tubule system, enabling interaction with local insulin receptors and, in turn, triggering local insulin signalling and local GLUT4 translocation. In parallel, we showed that the majority of GLUT4 depot vesicles do not move long distances but are depleted locally in the sarcolemma or t-tubule regions. Analysis of GLUT4 translocation in insulin-resistant muscle showed that, primarily, GLUT4 recruitment in the t-tubule region is affected. We have now analysed the kinetics of contraction-mediated GLUT4 translocation and reinternalization, as well as dilineated some of the key signalling points involved in these processes.