The study investigates the hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase (ACE2), a zinc metalloprotease closely related to angiotensin I-converting enzyme (ACE). ACE2 was purified and characterized, showing a pH optimum of 6.5 and enhanced activity in the presence of monovalent anions like Cl− and F−. Screening 126 biological peptides revealed that ACE2 hydrolyzes 11 peptides, primarily removing the C-terminal residue. High catalytic efficiency was observed for angiotensin II (1–8), apelin (1–3), and dynorphin A 1–13. The substrate specificity of ACE2 is influenced by the C-terminal sequence, with a consensus sequence of Pro-X1-3 residues-Pro-Hydrophobic. ACE2 also efficiently hydrolyzes des-Arg6-bradykinin but not bradykinin. The study suggests that ACE2 may play a role in the regulation of peptides involved in vasomotor control and pain modulation, such as angiotensin II, apelin, and dynorphin A 1–13.The study investigates the hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase (ACE2), a zinc metalloprotease closely related to angiotensin I-converting enzyme (ACE). ACE2 was purified and characterized, showing a pH optimum of 6.5 and enhanced activity in the presence of monovalent anions like Cl− and F−. Screening 126 biological peptides revealed that ACE2 hydrolyzes 11 peptides, primarily removing the C-terminal residue. High catalytic efficiency was observed for angiotensin II (1–8), apelin (1–3), and dynorphin A 1–13. The substrate specificity of ACE2 is influenced by the C-terminal sequence, with a consensus sequence of Pro-X1-3 residues-Pro-Hydrophobic. ACE2 also efficiently hydrolyzes des-Arg6-bradykinin but not bradykinin. The study suggests that ACE2 may play a role in the regulation of peptides involved in vasomotor control and pain modulation, such as angiotensin II, apelin, and dynorphin A 1–13.