We have investigated the subcellular distribution of GLUT4 by immunofluorescence microscopy after transfection with wild-type or mutant dynamin cDNA into Chinese hamster ovary cells expressing insulin receptor and GLUT4. In the basal state, GLUT4 was distributed exclusively within the cells in the cells overexpressing wild-type dynamin (CHOIR-GLUT4-WT) but was located at the cell surface in the cells overexpressing mutant dynamin (CHOIR-GLUT4-K44E). Insulin induced subcellular shift of GLUT4 to the cell surface in CHOIR-GLUT4-WT cells but had little effect in CHOIR-GLUT4-K44E cells. When insulin-stimulated cells were treated with wortmannin, GLUT4 was redistributed within the cells in CHOIR-GLUT4-WT cell, whereas it remained at the cell surface in CHOIR-GLUT4-K44E cell. These results suggest that dynamin is a regulatory GTPase in endocytosis of GLUT4.