Equilibrium [3H]cytochalasin B binding to class I sites of human red cell membranes (the sugar transporter) was examined in the presence and absence of intracellular or extracellular sugars known to interact with the transport system. D-Glucose, a transported sugar, is without effect on cytochalasin B binding when present in the extracellular medium but is an effective inhibitor of binding when present within the cell. Ethylidene glucose and maltose (reactive but nontransported sugars) inhibit cytochalasin B (CCB) binding when present either outside or inside the red cell. Inhibition by intracellular sugar (Si) is of the simple, linear competitive type. Inhibition by extracellular sugars (So) is more complex; the Kd(app) for cytochalasin B binding increases in a saturable fashion with [So]. These observations are compared with the predictions of the one-site, alternating conformer model and the two-site model for substrate binding to the sugar transporter, X. The experimental results are inconsistent with the one-site model but are explained by a two-site model in which the ternary complexes of So . X . Si or So . X . CCBi exist and where the binding sites for So and Si display negative cooperativity when occupied by nontransported substrate and little or no cooperativity when occupied by the transported species, D-glucose.