Hydrolysis of small peptides, like disaccharide hydrolysis, is an important function of the intestinal brush border, but little is known of the individual human peptidases. The purposes of this study were to detect all human brush border enzymes hydrolyzing dipeptides and tripeptides, identify the most discriminating substrate for each enzyme in order to permit assays in crude mixtures, and begin biochemical characterization of each enzyme. Four brush border peptidases were identified. Enzymes I (aspartate aminopeptidase, E.C. 3.4.11.7) and III (amino-oligopeptidase, E.C. 3.4.11.2) are known brush border enzymes. Enzymes II (membrane Gly-Leu peptidase) and IV (zinc stable Asp-Lys peptidase) have not been identified in human brush border previously. They are distinct from dipeptidyl aminopeptidase IV, carboxypeptidase, and gamma-glutamyl transferase. The substrate most discriminating for each enzyme is alpha-Glu-beta-naphthylamide for I (100% of the brush border activity for this substrate is due to enzyme I), glycylleucine for II (80%), leucyl-beta-naphthylamide for III (91%), and aspartyl-lysine in 5 mM Zn2+ for IV (63%). The enzymes are immunologically distinct and antibodies to each one localize to the brush border on immunohistochemical staining. Purification of 142-, 79-, 158-, and 46-fold was achieved for enzymes I through IV, respectively. Biochemical characteristics include slightly alkaline pH optima, molecular weights of 91,000-190,000, and evidence of metal ion involvement in activity. These studies provide necessary information for determining the role of brush border peptidase deficiencies in human disease.