Four facilitative glucose transporters isoforms, GLUT1/erythrocyte, GLUT2/liver, GLUT3/brain, and GLUT4/muscle-fat, as well as chimeric transporter proteins were expressed in Xenopus oocytes, and their properties were studied. The relative Km's of the transporters for 2-deoxyglucose were GLUT3 (Km = 1.8 mM) > GLUT4 (Km = 4.6 mM) > GLUT1 (Km = 6.9 mM) > GLUT2 (Km = 17.1 mM). In a similar fashion, the uptake of 2-deoxyglucose by GLUT1-, GLUT2-, and GLUT3-expressing oocytes was inhibited by a series of unlabeled hexoses and pentoses and by cytochalasin B in a similar hierarchical order. To determine if the functional unit of the glucose transporter was a monomer or higher-order multimer, the high-affinity transporter GLUT3 was coexpressed with either the low-affinity GLUT2 or a GLUT3 mutant which contained a transport inactivating Trp410-->Leu substitution. In oocytes expressing both GLUT2 and GLUT3, the transport activity associated with each transporter isoform could be distinguished kinetically. Similarly, there was no alteration in the kinetic parameters of GLUT3, or the ability of glucose or cytochalasin B to inhibit 2-deoxyglucose uptake, when coexpressed with up to a 3-fold greater amount of functionally inactive mutant of GLUT3. These studies suggest that the family of glucose transporters have similar binding sites which may be in the form of a functional monomeric unit when expressed in Xenopus oocytes.