Neutral metalloendopeptidase enzymes were identified and partially characterized in the brush-border membranes of rat small intestinal mucosal cells using insulin B chain and glutaryl-trialanine-4-methoxy-beta-naphthylamide as substrates. Three different molecular species of endopeptidase were identified by disc gel electrophoresis. These enzymes were shown to be distinct from pancreatic endopeptidases on the basis of the following: enrichment in the brush-border membrane fraction, site of hydrolysis of peptide substrates, sensitivity to specific proteinase inhibitors, and the presence of brush-border membrane-associated endopeptidase activity in mucosal cells of Thirty-Vella loops. Hydrolysis of the substrates was shown to be a two-step process involving initial cleavage by endopeptidase with secondary hydrolysis of the peptide products by brush-border membrane aminopeptidase N. Hydrolysis of both substrates was maximum at a neutral pH and was strongly inhibited by metal chelating agents, phosphoramidone, and amastatin. Intestinal perfusion studies using glutaryl-trialanine-4-methoxy-beta-naphthylamide suggest that these enzymes play a physiologic role in protein digestion. It was concluded that neutral endopeptidases are integral components of the intestinal brush-border membrane and work in concert with aminopeptidase N to hydrolyze dietary protein. This process may be of nutritional importance in normal subjects and those with diminished exocrine pancreatic function.