Glucose utilization and glycogen metabolism by human retinal pigment epithelium (RPE) cultures with high transepithelial resistance maintained on porous Millicell polycarbonate filters, were quantified by fluorophore-assisted carbohydrate electrophoresis (FACE). Glucose uptake was more efficient at the apical surface of the RPE. The utilization of glucose when restricted to either the apical or basal medium was also evaluated. Under both conditions, glucose was quickly transported to the opposite compartment and rapidly utilized. However, glucose from the apical compartment was depleted to a greater extent than from the basal compartment. The de novo synthesis and accumulation of glycogen accompanied glucose utilization. This was paralleled by a concomitant increase in lysosomal glycogen degradation measured as an increase in cell-associated maltodextrins. The highest levels of glucose in glycogen and maltodextrins occurred at 24 h, declining to basal levels at 72 h. Glucose transporter expression in the RPE cultures was evaluated with the reverse transcriptase-polymerase chain reaction. Glucose transporter-1 (GLUT 1) was the isoform expressed in these cells. GLUT 1 localization was determined by immunocytochemistry. GLUT 1 localizes to the apical and basolateral border of the RPE. The intensity of fluorescence was higher on the apical border. The rapid depletion of medium glucose suggests that RPE culture studies should replenish medium glucose more frequently than every 72 h to maintain physiologically relevant glucose concentrations. These studies are the first to demonstrate glucose, glycogen and maltodextrin metabolism by RPE cells, and their detection and quantitation by FACE.