We have expressed GLUT3 protein, an isoform of a facilitative glucose transporter, in Chinese hamster ovary cells by transfection of its cDNA using an expression vector. The expressed GLUT3 protein was detected by Western-blot analysis as a broad band of 45-65 kDa, indicating intensive glycosylation of the protein. The expressed GLUT3 protein was observed, by immunofluorescence staining, to be located mainly at the plasma membrane, and its expression was associated with a marked increase in glucose-transport activity. Kinetic analysis revealed that the K(m) value of GLUT3 protein for 3-O-methylglucose uptake was approx 35% of that of GLUT1 protein, whereas the K(m) value of GLUT3 protein for 2-deoxy-D-glucose uptake was very similar to that of GLUT1 protein. The V(max) value of GLUT3 protein for 3-O-methylglucose and 2-deoxyglucose uptake was approx. 20-50% of that of GLUT1 protein. GLUT3 protein was well photolabelled with [3H]cytochalasin B or a mannose derivative, 2-N-4-[3H](1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D- mannos-4-yloxy)-2-propylamine. Thus GLUT3 protein has very similar characteristics to GLUT1 protein including its subcellular localization, but exhibits lower K(m) and V(max) values for 3-O-methylglucose uptake.