Enkephalin-containing polypeptides derived from pro-enkephalin A, pro-enkephalin B, or pro-opiomelanocortin were inhibitors of enkephalin degradation by aminoenkephalinases purified from cytosol or membranes. Of the peptides, Argo-Met-enkephalin was the most potent inhibitor for the aminoenkephalinases, with an IC50 of about 0.6 microM, it was more effective than bestatin (IC50 = 0.8-1.0 microM). This inhibition was partly due to substrate competition. Argo-Met-enkephalin was hydrolyzed by aminoenkephalinases to form Arg, Tyr, and Gly-Gly-Phe-Met in a substrate-inhibited manner. The hexapeptide also inhibited the breakdown of Arg- and Tyr-beta-naphthylamide by the membrane aminoenkephalinase. Since Argo-Met-enkephalin did not inhibit leucine aminopeptidase, it was a more selective inhibitor than bestatin of Met-enkephalin breakdown by aminopeptidases. Argo-Met-enkephalin inhibited enkephalin breakdown by synaptosomal plasma membranes but not by brain slices. Our data suggest that in addition to their possible role as opioids, the enkephalin-containing polypeptides may be regulators of enkephalin levels.