This study utilized intestinal brush-border membrane vesicles obtained from organ donor intestine to characterize the absorption of D-glucose and L-leucine in the human intestine. Both D-glucose and L-leucine were taken up by sodium gradient-dependent active transport along the entire length of the small intestine. The relative magnitude of transport for both substrates under sodium gradient conditions followed the order distal jejunum greater than proximal jejunum greater than distal ileum. The number of carrier systems in these brush-border membrane vesicles was estimated by Eadie-Hofstee plot analysis. This analysis revealed that L-leucine was actively transported via a single high-affinity transport system for the length of the human small intestine. In contrast, the transport of D-glucose occurred via a high-affinity system along the length of the intestine and via a low-affinity, high-flux transport system that was limited to the proximal intestine. Both glucose transport systems were sodium dependent and phlorizin sensitive. The locations and apparent kinetic parameters of these transport systems indicated that these systems function efficiently in vivo as important mechanisms for carbohydrate and protein assimilation in humans. The presence of these active transport systems along the entire small intestine explains the formidable capacity for carbohydrate and protein assimilation in humans.