Equilibrative sugar uptake in human erythrocytes is characterized by a rapid phase, which equilibrates 66% of the cell water and by a slow phase which equilibrates 33% of the cell water. This behavior has been attributed to the preferential transport of -sugars by erythrocytes (Leitch, J. M., and A. Carruthers. 2007. ATP-dependent sugar transport complexity in human erythrocytes. Am J Physiol Cell Physiol S. 292: C974-86). The present study tests this hypothesis. The anomer theory requires that the relative compartment sizes of rapid and slow transport phases are determined by the proportions of - and -sugar in aqueous solution. This is observed with D-glucose and 3-O-methylglucose but not with 2-deoxy-D-glucose and D-mannose. The anomer hypothesis predicts that the slow transport phase, which represents -sugar transport, is eliminated when anomerization is accelerated to generate the more rapidly transported -sugar. Exogenous, intracellular mutarotase accelerates anomerization but has no effect on transport. The anomer hypothesis requires that transport inhibitors inhibit rapid and slow transport phases equally. This is observed with the endofacial site inhibitor cytochalasin B but not with the exofacial site inhibitors maltose or phloretin which inhibit only the rapid phase. Direct measurement of - and -sugar uptake demonstrates that erythrocytes transport - and -sugars with equal avidity. These findings refute the hypothesis that erythrocytes preferentially transport -sugars. We demonstrate that biphasic 3-O-methylglucose equilibrium exchange kinetics refute the simple carrier hypothesis for protein-mediated sugar transport but are compatible with a fixed-site transport mechanism regulated by intracellular ATP- and cell shape.