Depletion of ATP is known to inhibit glucose transport in human erythrocytes, but the kinetic mechanism of this effect is controversial. Selective ATP depletion of human erythrocytes by 10 micrograms/ml A23187 in the presence of extracellular calcium inhibited 3-O-methylglucose influx noncompetitively and efflux competitively. ATP depletion also decreased the ability of either equilibrated 3-O-methylglucose or extracellular maltose to inhibit cytochalasin B binding in intact cells, whereas neither total high-affinity cytochalasin B binding nor its Kd was affected. Under the one-site model of hexose transport these data indicate that ATP depletion decreases both the affinity of the inward-facing glucose carrier for substrate and its ability to reorient outwardly in intact cells.