After extracting converting enzyme from a membrane fraction of homogenized human kidney, "enkephalinase" activity was solubilized with Triton X-100. Ion-exchange chromatography resolved two peaks of the "enkephalinase" activity, both of which cleaved Leu5-enkephalin at the Gly3-Phe4 bond. The major "enkephalinase" form was purified 1140-fold to homogeneity with a 14% yield. This homogeneous "enkephalinase" had a specific activity of 46 mumol min-1 mg-1 with Leu5-enkephalin as substrate. The purified enzyme, in addition to hydrolyzing Leu5-enkephalin, cleaved synthetic substrates with protected N- and C-terminal ends. On the basis of the specificity of the enzyme and its inhibition by chelating agents, human "enkephalinase" can be classified as a neutral metalloendopeptidase with a broad substrate specificity. The activity of this neutral endopeptidase with several biologically active peptides was compared to that of homogeneous human kidney converting enzyme. Both enzymes inactivated bradykinin by release of the C-terminal dipeptide but were inhibited differentially by specific inhibitors. Comparison of hydrolysis of bradykinin with that of its protected C-terminal peptide indicated that the neutral endopeptidase is more active toward the larger substrate than is converting enzyme. Although the neutral endopeptidase did not convert angiotensin I to II, it did hydrolyze angiotensin I at Pro7-Phe8 and inactivate angiotensin II by cleavage at the Tyr4-Ile5 bond.