The interaction of the adipocyte/skeletal muscle glucose transporter (GLUT-4) with clathrin lattices may be important in maintaining its intracellular distribution. To better understand the role of clathrin lattices in the sorting of GLUT-4, we have attempted to determine the cellular origin of clathrin-coated vesicles (CCVs) that contain this transporter. The fungal toxin brefeldin A (BFA) causes the selective disassembly of clathrin lattices at the trans-Golgi network (TGN), but not at the plasma membrane (PM), thus providing a way of estimating the proportion of GLUT-4 in PM- versus TGN-derived clathrin lattices. Exposure of 3T3-L1 adipocytes to BFA resulted in a rapid disassembly of clathrin lattices at the TGN, observed by optical sectioning microscopy, and to a pronounced decrease in the yield of CCVs purified from these cells. Thus, CCVs isolated from BFA-treated cells are likely to be derived from the PM. Immunoblotting experiments revealed the presence of GLUT-4 in such CCVs, suggesting that under basal conditions the transporter is continually retrieved from the PM through the CCV pathway. Exposure of both BFA-treated or non-treated cells to insulin resulted in a 4-6-fold increase in the concentration of GLUT-4 at the PM. In parallel, the concentration of GLUT-4 in PM-derived CCVs decreased by 60%. These results suggest (a) that the effect of insulin to increase the cell surface concentration of GLUT-4 is not inhibited by BFA, and (b) that a decreased association of GLUT-4 with endocytic CCVs may be important in facilitating its increased cell surface concentration in response to the hormone.