Insulin and guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) both stimulate glucose transport and translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma membrane in adipocytes. Previous studies suggest that these effects may be mediated by different mechanisms. In this study we have tested the hypothesis that these agonists recruit GLUT4 by distinct trafficking mechanisms, possibly involving mobilization of distinct intracellular compartments. We show that ablation of the endosomal system using transferrin-HRP causes a modest inhibition ( approximately 30%) of insulin-stimulated GLUT4 translocation. In contrast, the GTPgammaS response was significantly attenuated ( approximately 85%) under the same conditions. Introduction of a GST fusion protein encompassing the cytosolic tail of the v-SNARE cellubrevin inhibited GTPgammaS-stimulated GLUT4 translocation by approximately 40% but had no effect on the insulin response. Conversely, a fusion protein encompassing the cytosolic tail of vesicle-associated membrane protein-2 had no significant effect on GTPgammaS-stimulated GLUT4 translocation but inhibited the insulin response by approximately 40%. GTPgammaS- and insulin-stimulated GLUT1 translocation were both partially inhibited by GST-cellubrevin ( approximately 50%) but not by GST-vesicle-associated membrane protein-2. Incubation of streptolysin O-permeabilized 3T3-L1 adipocytes with GTPgammaS caused a marked accumulation of Rab4 and Rab5 at the cell surface, whereas other Rab proteins (Rab7 and Rab11) were unaffected. These data are consistent with the localization of GLUT4 to two distinct intracellular compartments from which it can move to the cell surface independently using distinct sets of trafficking molecules.