Exercise stimulates glucose uptake into skeletal muscle, through the redistribution of GLUT4 from intracellular vesicles to the sarcolemma. The underlying mechanism is still under investigation, and our recent studies show that ATP is an autocrine mediator of this response1. How ATP would contribute to GLUT4 translocation is unknown. Insulin also stimulates glucose uptake and this involves activation of the Rac1 GTPase and dynamic rearrangement of actin-filaments, in addition to stimulating the canonical Akt pathway and downstream Rab GTPases2. Here we investigate if extracellular ATP induces cortical actin remodeling and if this occurs through Rho activation, leading to GLUT4 insertion into the cell surface. Material and Methods: L6 myoblasts and myotubes stably expressing GLUT4myc were used as readout, by stimulation with, ATP (100 µM, 5 min) or insulin (100 nM, 5 min). Actin filaments were visualized by confocal fluorescence microscopy using Alexa488-conjugated phalloidin. Rac and Cdc42 activation was assessed by GELISA. L6 muscle cells were also pretreated with latrunculin A, or siRNA to Rac1 prior to stimulation as above followed by assessment cell-surface GLUT4myc quantification. Results: Extracellular ATP and insulin each promoted rapid GLUT4 translocation and insertion of GLUT4 into the membrane of L6 GLUT4myc myoblasts. Extracellular ATP rapidly activated GTP-loading onto Rac1 and Cdc42. ATP-induced translocation of GLUT4 was partially blocked by silencing Rac1 or Cdc42. Work in progress is assessing the role of the actin cytoskeleton in the response to ATP. This work suggests that myotube contraction leads to autocrine secretion of factors including ATP that contribute to GLUT4 mobilization, via an intracellular signalling pathway involving Rac1 and Cdc42.