In the mammalian digestive tract, small peptides are absorbed by a H⁺-coupled peptide transport system. Using an antibody against the rat H⁺/peptide constransporter (PepT1), we examined the localization of PepT1 immunohistochemically along the rat digestive tract. PepT1 was detected in the small intestine (duodenum, jejunum, and ileum), but not in the esophagus, stomach, colon, or rectum. PepT1 was especially enriched in the villi, where it was localized at the brush border of the absorptive epithelial cells. PepT1 was not detected in the mucus-secreting goblet cells or less-differentiated epithelial cells in the crypts. These observations show that PepT1 is specific to the brush border of the differentiated absorptive epithelial cells and suggest that H⁺-coupled uptake of small peptides and peptide-like drugs occurs at the apical membrane of these cells in the small intestine.