Human red cell membrane vesicles stripped of peripheral proteins and proteoliposomes with reconstituted red cell glucose transporter (Glut1) were sterically immobilized in gel beads by freezethawing. The specific interactions between the transport inhibitor cytochalasin B (CB), D-glucose, and Glut1 were analyzed by quantitative frontal affinity chromatography. The dissociation constants, Kd(CB), for the interaction between CB and Glut1 in vesicles and proteoliposomes were similar, the average value being 92 +/- 5 nM at an ionic strength I of 0.05. Kd(CB) for Glut1 in vesicles decreased with increasing ionic strength to become 46 nM at I = 0.5. The affinity of glucose was significantly higher for Glut1 in vesicles (Kd = 24 +/- 2 mM) than for reconstituted Glut1 (Kd = 37 +/- 2 mM). The frontal analysis allowed determination of the amount of CB binding sites, which was found to be 0.33 +/- 0.06 mol per mole of Glut1 monomer (Mr = 54 000). The CB binding capacity of Glut1 in the vesicles and the proteoliposomes was stable in the presence of dithioerythritol over periods of several weeks at room temperature.