Insulin stimulates translocation of GLUT4 from an intracellular compartment to the plasma membrane in adipocytes. As a significant amount of GLUT4 is localised to the TGN, independently of the biosynthetic pathway, one possibility is that trafficking via the TGN is important in either intracellular sequestration or insulin-dependent movement to the cell surface. In this study we have used immuno-electron microscopy to show that GLUT4 is localised to AP-1 vesicles in the TGN region in 3T3-L1 adipocytes. To dissect the role of this trafficking pathway we used brefeldin A (BFA) to disrupt AP-1 association with membranes. Despite a reorganisation of GLUT4 compartments following BFA treatment, the intracellular sequestration of GLUT4, and its insulin-dependent movement to the cell surface, was unaffected. BFA increased the half time of reversal of insulin-stimulated glucose transport from 17 to 30 min but did not prevent complete reversal. Furthermore, following reversal restimulation of glucose transport activity by insulin was not compromised. We conclude that under basal conditions GLUT4 cycles between the TGN and endosomes via the AP-1 pathway. However, neither this pathway, nor any other BFA-sensitive pathway, appears to play a major role in insulin-dependent recruitment of GLUT4 to the cell surface.