In this study, we have characterized the Naiglucose transporter in polarized monolayers formed by the clonal human colon carcinoma cell line HT-29-D4. Isotopic tracer flux measurements show that differentiated HT-29-D4 cells possess a sodium-dependent a-methyl-D-glucopyranoside (AMG) uptake that is competed for by increasing concentrations of D-glucose, D-galactose, and phlorizin. This transport is exclusively localized on the apical side of the epithelium. Kinetic data demonstrate the existence of a single Michaelian sodium-dependent AMG transporter with a Km of 1.2 +-0.12 m M and a Vmax of 3.24 +-0.25 nmolimg of protein per min. Hill analysis reveals a coefficient of 1.9 ? 0.03, consistent with at least two sodium ions involved in AMG transport. Interestingly, the cotransporter function is not modulated by glucose in the culture medium. Transepithelial electrical parameter measurements show that the transepithelial potential difference (Vt) is glucose dependent and phlorizin sensitive. Antibodies directed against a peptide of the rabbit intestinal glucose cotransporter (Ser402-Lys420) recognize, in western blot experiments, the characteristic bands of the cotransporter on a crude membrane preparation of differentiated HT-29-D4 cells and react strongly with the apical domain of the monolayer in immunofluorescence experiments. We conclude that HT-29-D4 cells express the sodiumiglucose cotransporter SGLTl at their apical membrane and that this transporter generates the basal transepithelial potential difference.