Previous studies from our laboratory have characterized the transport process of biotin across the brush border membrane and the basolateral membrane of the human intestine. In this study we further characterized biotin transport in the human intestine by examining the vitamin's transport process in different areas of the small intestine (duodenum, jejunum, and ileum) and the effect of pH on the transport process using a brush border membrane vesicle technique. In all areas examined, the transport of biotin as a function of concentration was saturable in the presence of a Na+ gradient (out greater than in) but was linear and lower in the presence of a choline gradient (out greater than in). Transport of biotin by the Na+-dependent process (i.e., the carrier-mediated process) was found to be higher in the duodenum than the jejunum, which was in turn higher than that in the ileum. This decrease in biotin transport distally was found to be due to a decrease in the Vmax of the transport process of the vitamin with no changes in the apparent Km. This indicates that the number (i.e., the density) of transport carriers for biotin decreases distally. In the presence of a Na+ gradient (out greater than in), decreasing incubation buffer pH from 8.0 to 5.5 (intravesicular pH was 7.4) was found to cause an increase in biotin transport. This increase was found to be due to the acidic buffer pH (i.e., not due to the pH gradient imposed across the membrane) and occurred through an increase in the transport of the vitamin by the nonmediated process. These results demonstrate that the proximal part of the small intestine is the site of maximum transport of biotin in humans. Furthermore, variation in incubation medium pH affects biotin transport through changes in the substrate transport by the nonmediated process.