We have expressed the human isoforms of the liver-type (GLUT2) and brain-type (GLUT3) facilitative glucose transporters in oocytes from Xenopus laevis via injection of in vitro transcribed mRNA. As reported previously [Gould, Thomas, Jess and Bell (1991) Biochemistry 30, 5139-5145], GLUT2 mediates the transport of fructose and galactose, and GLUT3 mediates the transport of galactose. We have examined the effects of D-glucose, D-fructose and maltose on deoxyglucose transport in oocytes expressing GLUT2, and D-glucose, D-galactose and maltose on deoxyglucose transport in oocytes expressing GLUT3, and show that each sugar is a competitive inhibitor of transport. Moreover, D-glucose and maltose competitively inhibit fructose transport by GLUT2 and galactose transport by GLUT3, indicating that the transport of the alternative substrates for these transporters is likely to be mediated by the same outward-facing sugar-binding site used by glucose. Cytochalasin B is a non-competitive inhibitor of glucose transport by the well-characterized GLUT1 isoform. We show here that cytochalasin B is also a non-competitive inhibitor of the transport of deoxyglucose and alternative substrates by GLUT2 and GLUT3 expressed in oocytes. Km and Ki values for each substrate and inhibitor are presented for each isoform, together with further analysis of the binding sites for alternative substrates for these transporter isoforms.