The present study was conducted to quantify and characterize the metabolism of circulating low mol. weight opioid peptides by human blood plasma and vascular endothelium. Anal. of [Met5]- and [Leu5]-enkephalin metabolism by human plasma established rapid degradation (32 nmol/min/mL), primarily (>95%) due to N-terminal hydrolysis by aminopeptidase M (AmM; EC 3.4.11.2). In contrast, the combined actions of plasma angiotensin converting enzyme (ACE; EC 3.4.15.1) and "enkephalinase"/neutral endopeptidase-24.11 (NEP; EC 3.4.24.11) accounted for <5% of enkephalin degradation (1.0 nmol/min/mL). Plasma carboxypeptidase N (CPN; EC 3.4.17.3) converted [Leu5, Arg6]enkephalin to [Leu5]-enkephalin (10 nmol/min/mL), but AmM-mediated degradation was nearly 3-fold more rapid (26 nmol/min/mL). Among C-terminally extended [Leu5]enkephalins, the rates of plasma AmM-mediated degradation decreased with increasing peptide length. Hydrolysis of dynorphin A, containing 17 residues, was 1/20 that of [Leu5]-enkephalin. As found for plasma, [Met5]enkephalin degradation by cultured human endothelium (0.33 nmol/min/well) was primarily (>80%) due to AmM (0.28 nmol/min/well) rather than ACE (0.05 nmol/min/well). These data are consistent with an important role for plasma and endothelial AmM in terminating the actions of circulating low mol. weight enkephalins in humans circulation. Thus, the structures of agents that will be used as opioid receptor stimulants must be modified to ensure resistance to metabolism by aminopeptidase M.