The water-soluble vitamin B6 (pyridoxine) is important for normal cellular functions, growth and development. The vitamin is obtained from two exogenous sources: a dietary source, which is absorbed in the small intestine, and a bacterial source where the vitamin is synthesized in significant quantities by the normal microflora of the large intestine. Evidence exists to suggest the bioavailability of the later source of the vitamin but nothing is known about the mechanism involved and its regulation. In this study we addressed these issues using young adult mouse colonic epithelial (YAMC) cells, and human colonic apical membrane vesicles (AMV) as models, and using ³H-pyridoxine as the uptake substrate. The results showed the initial rate of ³H-pyridoxine uptake by YAMC cells to be: 1) energy- and temperature- (but not Na-) dependent and occur without metabolic alteration in the transported substrate; 2) saturable as a function of concentration with an apparent Km and Vmax of 2.1 ± 0.5 µM and 53.4 ± 4.3 pmol/mg protein/3min, respectively; 3) cis-inhibited by unlabeled pyridoxine and its structural analogs, but not by the unrelated compounds theophylline, penicillamine and isoniazid; 4) transstimulated by unlabeled pyridoxine; 5) amiloride-sensitive and 6) regulated by extracellular and intracellular factors. Uptake of pyridoxine by native human colonic AMV was also found to involve a carrier-mediated process. These studies demonstrate, for the first time, the functional existence of a specific and regulatable carrier-mediated process for pyridoxine uptake by mammalian colonocytes.