One of the primary inactivating cleavages of neurotensin (NT) by rat brain synaptic membranes occurs at the Arg8-Arg9 peptide bond, leading to the formation of NT1-8 and NT9-13. The involvement at this site of a recently purified metalloendopeptidase was demonstrated by the use of its specific inhibitor, N-[1(R,S)-carboxy-2-phenylethyl]-alanylalanylphenylalanine-p-amino -benzoate, which exerts an inhibition on NT1-8 formation with an IC50 (0.6 microM) close to its Ki for the purified metalloendopeptidase (1.94 microM). Furthermore, we established the role of a postproline dipeptidyl-aminopeptidase in the secondary processing of NT9-13 formation.