A membrane-bound neutral endopeptidase which hydrolyzes Suc-(Ala)3-pNA to succinyl dialanine and Ala-pNA has been purified from human placenta. The enzyme was solubilized from membranes with DOC and papain, and was purified about 5000-fold by successive chromatographies on Sephadex G-200, DEAE-Sephacel, butyl-Toyopearl 650, and Sephacryl S-300. It was found to be homogeneous on SDS-polyacrylamide gel electrophoresis and to have a molecular weight of about 70,000. It was strongly inhibited by phosphoramidon, thiorphan, and metal-chelating agents, but was not affected by most other protease inhibitors. These findings indicate that it can be classified as a phosphoramidon-sensitive neutral endopeptidase. With biologically active peptides as substrates, the enzyme preferentially cleaved the bonds at the amino side of hydrophobic amino acid residues. The physiological significance of this enzyme is discussed with reference to the placental barrier.