In adipose cells, insulin induces the translocation of GLUT4 by stimulating their exocytosis from a basal intracellular compartment to the plasma membrane. Increasing overexpression of a hemagglutinin (HA) epitope-tagged GLUT4 in rat adipose cells results in a roughly proportional increase in cell surface HA-GLUT4 levels in the basal state, accompanied by a marked reduction of the fold HA-GLUT4 translocation in response to insulin. Using biochemical methods and cotransfection experiments with differently epitope-tagged GLUT4, we show that overexpression of GLUT4 does not affect the intracellular sequestration of GLUT4 in the absence of insulin, but rather reduces the relative insulin-stimulated GLUT4 translocation to the plasma membrane. In contrast, overexpression of GLUT1 does not interfere with the targeting of GLUT4 and vice versa. These results suggest that the mechanism involved in the intracellular sequestration of GLUT4 has a high capacity whereas the mechanism for GLUT4 translocation is readily saturated by overexpression of GLUT4, implicating an active translocation machinery in the exocytosis of GLUT4.