Biotin transport was studied using brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex. An inwardly directed Na+ gradient stimulated biotin uptake into brush-border membrane vesicles and a transient accumulation of the anion against its concentration gradient was observed. In contrast, uptake of biotin by basolateral membrane vesicles was found to be Na+-gradient insensitive. Generation of a negative intravesicular potential by valinomycin-induced K+ diffusion potentials or by the presence of Na+ salts of anions of different permeabilities enhanced biotin uptake by brush-border membrane vesicles, suggesting an electrogenic mechanism. The Na+ gradient-dependent uptake of biotin into brush-border membrane vesicles was saturable with an apparent K m of 28 μM. The Na+-dependent uptake of tracer biotin was significantly inhibited by 50μM biotin, and thioetic acid but not by 50 μM l-lactate, d-glucose, or succinate. Finally, the existence in both types of membrane vesicles of a H+/biotin cotransport system could not be demonstrated. These results are consistent with a model for biotin reabsorption in which the Na+/biotin− cotransporter in luminal membranes provides the driving force for uphill transport of this vitamin.