Discriminating substrates were used to develop assays that accurately measure the activity of each of four brush border peptidases in peroral jejunal biopsy specimens. Seventy-five biopsy specimens, 43 normal and 32 abnormal, were assayed for aminopeptidase A (EC 3.4.11.7), aminopeptidase N (EC 3.4.11.2), and disaccharidases, and 37 of these were assayed for membrane glycylleucine peptidase and Zn2+-stable aspartyllysine peptidase. Peptidase levels in normal biopsy specimens from males and females did not differ. Only aminopeptidase A levels changed significantly with age. The mean +/- SD units per gram of protein for each enzyme in peroral biopsy specimens of normal intestine were aminopeptidase A 1.7 +/- 0.7 for children and 3.1 +/- 1.3 for adults, membrane glycylleucine peptidase 16.3 +/- 10.6, aminopeptidase N 35.5 +/- 13.2, and Zn2+-stable aspartyllysine peptidase 42.6 +/- 28.5. In abnormal biopsy specimens, levels of three of the peptidases and the disaccharidases were significantly decreased, 37% to 51%. In contrast, aminopeptidase N activity was decreased only 7% in the abnormal biopsy specimens. These results provide a basis for further studies investigating the role of brush border peptidases in normal digestion and various pathologic states, including the search for individuals with brush border peptidase deficiencies.