Little is known of biotin handling by transporting epithelium. Accordingly, we have examined the characteristics of biotin uptake by rat renal tubular epithelium. Renal cortical slices showed concentrative, temperature-sensitive uptake of biotin. Renal brushborder membrane vesicles exhibited an "overshoot" phenomenon with uptake of 1.9 nM biotin in the presence of a 100 mM NaCl gradient. This overshoot was reduced in magnitude with reduction of the sodium gradient to 50 mM. Biocytin significantly reduced uptake by the vesicles. Concentration-dependent studies yielded an apparent transport Km of 200 nM. We conclude that biotin is actively transported by the rat renal proximal tubule by a system which is at least partially Na+ dependent, and shared by biocytin.