The recently discovered native endomorphins play an important role in opioid analgesia, but their metabolic fate in the organism remains relatively little known. This paper describes the application of high-performance liquid chromatography combined with electrospray ionization mass spectrometry to identify the degradation products resulting from the incubation of endomorphins with proteolytic enzymes. The native endomorphin-1, H-Tyr-Pro-Trp-Phe-NH 2 (1), and endomorphin-2, H-Tyr-Pro-Phe-Phe-NH 2 (2), and an analog of endomorphin-2, H-Tyr-Pro-Phe-Phe-OH (3), were synthetized, and the levels of their resistance against carboxypeptidase A, carboxypeptidase Y, aminopeptidase M and proteinase A were determined. The patterns of peptide metabolites identified by this method indicated that carboxypeptidase Y first hydrolyzes the C-terminal amide group to a carboxy group, and then splits the peptides at the Trp 3 -Phe 4 or Phe 3 -Phe 4 bond. The remaining fragment peptides are stable against the enzymes investigated. Carboxypeptidase A degrades only analog 3 at the Phe 3 -Phe 4 bond. Aminopeptidase M cleaves the peptides at the Pro 2 -Trp 3 or Pro 2 -Phe 3 bond. The C-terminal fragments hydrolyze further, giving amino acids and Phe-NH 2 -s while the N-terminal part displays a resistance to further aminopeptidase M digestion. Proteinase A exhibits a similar effect to carboxypeptidase Y: the C-terminal amide group is first converted to a carboxy group, and one amino acid is then split off from the C-terminal side.