The molecular signals and glucose transporter-4 (GLUT4) dynamics associated with exercise/contraction-stimulated glucose transport into skeletal muscle are not completely understood. The trigger to muscle contraction is membrane depolarization. Therefore, we explored the molecular mechanisms and GLUT4 traffic properties participating in K+ depolarization-stimulated glucose transport using L6-GLUT4myc cells. We observed that Ca2+ chelators and inhibitors to conventional PKC prevented K+ depolarization-induced effects. We further observed that depolarization largely reduces GLUT4 internalization. Lack of a contractile apparatus in L6-GLUT4myc cells requires us to study contraction-mediated glucose transport in intact skeletal muscle. The impasse is the lack of an accurate method for measuring surface GLUT4 in mature tissue. Therefore, we attempted to establish a biochemical assay using transgenic mice expressing GLUT4myc in skeletal muscle. Although the proposed carry-over assay efficiently detected GLUT4 translocation in L6-GLUT4myc cells, further modifications are required before insulin-stimulated GLUT4 translocation can be successfully measured in isolated GLUT4myc skeletal muscle.