Transport of biotin across human intestinal brush-border membrane (BBM) was examined using brush-border membrane vesicle (BBMV) technique. Uptake of biotin by BBMV is mostly the result of transport of the substrate into an active intravesicular space with little binding to membrane surfaces. The transport of biotin was carrier mediated and was 1) Na+ (but not K+) gradient dependent with a distinct "over-shoot" phenomenon, 2) saturable as a function of concentration in the presence of a Na+ (but not a K+) gradient with an apparent Km and Vmax for the Na+ gradient-dependent system of 5.26 microM and 13.47 pmol.mg protein-1.20 s-1, respectively, and 3) inhibited by structural analogues and related compounds. Unlike the electrogenic transport of D-glucose, transport of the anionic biotin in the presence of a Na+ gradient (out greater than in) was not affected by imposing a relatively positive intravesicular electrical potential, suggesting that biotin transport is most likely an electroneutral process. These results demonstrate the existence of a carrier-mediated system for biotin transport in human BBM and show that the transport process is Na+ gradient dependent and electrically silent. It is suggested that biotin transport across the BBM is driven by a Na+ gradient most probably through a biotin-Na+ cotransport system.