We have characterized the insulin-dependent increase in glucose transport in human adipocytes using subcellular fractionation and antibodies specific for the two isoforms of the glucose transporter that are expressed in these cells. Plasma membranes isolated from untreated human fat cells contain the erythroid/GLUT1 isoform of the glucose transporter almost exclusively whereas the muscle-fat/GLUT4 transporter isoform is most abundant in intracellular microsomal membranes in resting cells. After exposure of adipocytes to insulin, the muscle-fat isoform is dramatically increased in the plasma membrane whereas the erythroid isoform barely changes in response to insulin. Thus, the total insulin-mediated increase in plasma membrane glucose transporters, confirmed by affinity labeling of both transporter isoforms, must be due to the increase in the muscle-fat/GLUT4 transporter. The two isoforms exist in different vesicle populations as shown by immunoadsorption of the muscle fat isoform-containing vesicles which are essentially devoid of the erythroid transporter. These data indicate that the insulin-mediated increases in glucose transport in human fat cells is a result of the translocation of vesicles uniquely containing the muscle-fat glucose transporter isoform.