It is well established that insulin stimulation of glucose uptake requires the translocation of intracellular localized GLUT4 protein to the cell surface membrane. This plasma membrane-redistributed GLUT4 protein was partially co-localized with caveolin as determined by confocal fluorescent microscopy but was fully excluded from lipid rafts based upon Triton X-100 extractability. Cholesterol depletion with methyl-beta-cyclodextrin, filipin, or cholesterol oxidase resulted in an insulin-independent increase in the amount of plasma membrane-localized GLUT4 that was fully reversible by cholesterol replenishment. This basal accumulation of cell surface GLUT4 occurred due to an inhibition of GLUT4 endocytosis. However, this effect was not specific since cholesterol extraction also resulted in a dramatic inhibition of clathrin-mediated endocytosis as assessed by transferrin receptor internalization. To functionally distinguish between caveolin- and clathrin-dependent endocytic processes, we took advantage of a dominant-interfering caveolin 1 mutant (Cav1/S80E) that specifically disrupts caveolae organization. Expression of Cav1/S80E, but not the wild type (Cav1/WT) or Cav1/S80A mutant, inhibited cholera toxin B internalization without any significant effect on transferrin receptor endocytosis. In parallel, Cav1/S80E expression increased the amount of plasma membrane-localized GLUT4 protein in an insulin-independent manner. Although Cav1/S80E also decreased GLUT4 endocytosis, the extent of GLUT4 internalization was only partially reduced ( approximately 40%). In addition, expression of Cav1/WT and Cav1/S80A enhanced GLUT4 endocytosis by approximately 20%. Together, these data indicate that the endocytosis of GLUT4 requires clathrin-mediated endocytosis but that the higher order structural organization of plasma membrane caveolin has a significant influence on this process.