D-Glucose uptake in isolated rabbit enterocytes was studied using confocal microscopy and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NDBG), a D-glucose fluorescent analogue, by analysing the fluorescence of apical and basal enterocyte zones. Under normal conditions, apical fluorescence was always higher than basal, presumably due to the location of the Na+-D-glucose cotransporter in the brush-border membrane. After blocking this transporter with phlorizin, apical and basal fluorescence values were similar. This suggests that both brush-border and basolateral membranes participate in phlorizin-insensitive D-glucose transport, since transport across only one membrane cannot explain the uniform overall fluorescence observed. Similarly, after inhibiting the Na+-D-glucose cotransporter by incubating the enterocytes in a medium containing 0.5 mM Na+, neither apical nor basal fluorescence predominated. In contrast, with 130.5 mM extracellular Na+, apical fluorescence was clearly higher than basal fluorescence. These results suggest that phlorizin-insensitive, Na+-independent 2-NDBG uptake occurred through both brush-border and basolateral membranes, probably via the glucose uniporters GLUT2 and GLUT5, suggesting that the latter is a D-glucose transporter.