1. The relative rates of hydrolysis of a series of glycine homopeptides by guinea-pig small intestinal mucosa and by isolated brush borders have been studied. Oligopeptides up to and including hexaglycine were hydrolysed. No activity was detected against a series of homopolypeptides of molecular weight 10000–100000.
2. Except for activity against diglycine, the peptidase activity was greater in the brush-border fraction than in the original homogenate. The relative activity of the peptidase in the brush borders compared to the homogenate increased with increasing length oligopeptide substrate.
3. Analysis of the enzyme reaction products indicated that the brush border contained an exopeptidase. Studies with peptide derivatives containing N- and C-terminal blocking groups and with tetraglycine synthesized with either the N- or C-terminal residues labelled, demonstrated that the brush borders contained an amino-oligopeptidase.
4. Studies with inhibitors and with purified gastric and pancreatic enzymes indicated that this glycine oligopeptidase activity was not due to enzymes from the intestinal lumen which had been adsorbed to the brush borders.