Enkephalins can be degraded by a variety of peptidases. We have characterized several membrane-associated brain peptidases in an effort to determine which if any are concerned with the physiological inactivation of synaptically released enkephalin. We have distinguished two carboxyl-directed dipeptidylpeptidases, designated enkephalinase A1 and A2, that give rise to the Tyr-Gly-Gly fragment. Both enzymes are physically separable from angiotensin converting enzyme. Regional variations in enkephalinase A1 activity and opiate receptors are similar. A novel amino-terminal-directed dipeptidylpeptidase, enkephalinase B, which generates Tyr-Gly, has been identified. All of these enzymes as well as aminopeptidase have been solubilized from brain membranes by detergent treatment and have been mutually resolved by DEAE column chromatography. Enkephalinase A1 has been purified 1500-fold, to apparent homogeneity.