A recent study by C.F. Burant et al. (13) demonstrates that GLUT5 is a high-affinity fructose transporter with a much lower capacity to transport glucose. To characterize the potential role of GLUT5 in fructose and glucose transport in insulin-sensitive tissues, we investigated the distribution and insulin-stimulated translocation of the GLUT5 protein in human tissues by immunoblotting with an antibody to the COOH-terminus of the human GLUT5 sequence. GLUT5 was detected in postnuclear membranes from the small intestine, kidney, heart, four different skeletal muscle groups, and the brain, and in plasma membranes from adipocytes. Cytochalasin-B photolabeled a 53,000-M(r) protein in small intestine membranes that was immunoprecipitated by the GLUT5 antibody; labeling was inhibited by D- but not L-glucose. N-glycanase treatment resulted in a band of 45,000 M(r) in all tissues. Plasma membranes were prepared from isolated adipocytes from 5 nonobese and 4 obese subjects. Incubation of adipocytes from either group with 7 nM insulin did not recruit GLUT5 to the plasma membrane, in spite of a 54% insulin-stimulated increase in GLUT4 in nonobese subjects. Thus, GLUT5 appears to be a constitutive sugar transporter that is expressed in many tissues. Further studies are needed to define its overall contribution to fructose and glucose transport in insulin-responsive tissues and brain.