The substrate specificity of duodenase from bovine duodenum mucosa to synthetic and natural polypeptides was studied. Amino acid residues preferential for duodenase in the P1 and P2 positions of the substrate were determined. It was shown that the enzyme is synthesized in epithelial secretory cells of duodenal (Brunner's) glands and enters, as part of the secreta, into the lumen of the duodenum. The possible role of duodenase as an activator of proenteropeptidase is discussed.