A Na+-dependent hexose transport system with similar characteristics that observed in the kidney is retained in a cultured epithelial cell line from pig kidney (LLC-PK1). The active transport of oc methyl-D-glucoside (oc MGP), a nonmetabolizable sugar, which shares the glucose-galactose transport system in kidney cells is mediated through a Na+-dependent, substrate-saturable process. The kinetic analysis of the effect of Na+ on the uptake of ocMGP indicated that the Na+-sugar cotransport system is an affinity type system in which the binding of either sugar or Na+ carrier increases the affinity for the other ligand without affecting the Vmax. The sequence of selectivity for different sugars studied by the inhibition produced in the uptake of ocMGP is very similar to that reported in rat kidney, rabbit kidney cortex slices, and rabbit renal brush border membrane vesicles. Phlorizin, even at very low concentration, almost completely inhibits ocMGP uptake. Conversely, phloretin at the same low concentration stimulated the sugar accumulation by inhibition of efflux, probably at the level of the basolateral membrane. Sulfhydryl group inhibitors also blocked the ocMGP uptake, suggesting that these groups were required for normal functioning of the sugar carrier system. This sugar transport system is an important functional marker to study the molecular events associated with the development of polarization in epithelial cells.