In order to elucidate the mechanism by which the electrochemical Na+ gradient energizes glucose transport, the energy-dependence of high affinity phlorizin binding to isolated renal microvillus membrane vesicles was examined. Phlorizin is a competitive inhibitor of glucose transport but is not itself transported. Extravesicular Na+ accelerated the rate of phlorizin binding and inhibited the rate of dissociation of bound glycoside. Maneuvers to enhance intravesicular electronegativity stimulated phlorizin uptake and those to enhance intravesicular electronegativity stimulated phlorizin uptake and those to enhance intravesicular electropositivity inhibited. However, alterations in electrical potential were without effect on the rate release of bound phlorizin. Intravesicular Na+ inhibited the phlorizin uptake rate. The results are consistent with a model of the glucose transporter in which (i) Na+ increases the binding affinity of the carrier, (ii) the free carrier is negatively charged, and (iii) the translocation of the carrier is inhibited by the binding of Na+ in the absence of sugar. The electrochemical Na+ gradient thus energizes both glucose transport and phlorizin binding through its effect on the affinity and appearance of the free carrier at the membrane surface rather than through an effect on sugar translocation per se.