OBJECTIVE A detailed investigation of the metabolic routes and rates of Dyn A1-13 in human blood and plasma was performed. METHODS Human plasma was incubated at 37 degrees C with dynorphin A 1-13 (Dyn A1-13, 15-20 microM). The generated dynorphin fragments were separated by a new ion-pair chromatographic method and identified by matrix assisted laser desorption mass spectroscopy. The kinetic behavior of parent compound and metabolites was evaluated in the absence and presence of enzyme inhibitors. RESULTS The major plasma metabolites of Dyn A1-13 were Dyn A1-12, A2-12, A4-12 and A4-8. Further metabolites were Dyn A2-13, A3-13, A3-12, A5-12, A6-12, A7-12, A1-10, A2-10, A2-8 and A3-8. At 37 degrees C, Dyn A1-13 had a half-life of less than one minute in plasma and blood. Plasma half-lives of major metabolites ranged between 0.5 and 4 min. Inter- and intra-individual differences in healthy volunteers were 30% (c.v.). Dyn A1-13 is mainly metabolized by carboxypeptidases to Dyn A1-12 (80%) and by aminopeptidases to Dyn A2-13 (15%). Dyn A1-12 and Dyn A2-13 are predominantly converted into Dyn A2-12 (67% of Dyn A1-13). Subsequent metabolic steps yield Dyn A3-12 (16%), Dyn A4-12 (37%) and Dyn A4-8 (33%). Aminopeptidases generate Dyn A2-12, A3-12, A4-12, A5-12. ACE metabolizes Dyn A1-12 (19%), A2-12 (33%), A3-12 (34%) and A4-12 (46%). Bestatin-sensitive endopeptidases (possibly endopeptidase 24.11) metabolize 30% of Dyn A2-12. Dyn A4-8 is formed via Dyn A4-12 (23% of Dyn A4-12) and Dyn A2-10 (37% of Dyn A2-10). CONCLUSIONS The combination of enzyme inhibition experiments and noncompartmental kinetic analysis proved to be a powerful tool for the detailed evaluation of the metabolic fate of Dyn A1-13 in human blood and plasma.