Chronic imbalance between production and degradation of the human amyloid-beta peptide (Aβ) is assumed to play an important role in pathogenesis of Alzheimer's disease (AD). Post-translational modifications of Aβ could influence its interactions with specifically cleaving proteases and, therefore, perturb the Aβ homeostasis. The angiotensin-converting enzyme (ACE) was previously shown to degrade non-modified Aβ in vitro and in cells. In the presented work, we investigated the effect of isomerization of Asp-7, a common non-enzymatic age-related modification found in AD-associated Aβ species, on hydrolysis of Aβ by ACE. Two synthetic peptides corresponding to the Aβ region 1-16 with either Asp or isoAsp residues in position 7 were examined as monomelic soluble substrates for the N- as well as for the C-domain of ACE. The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) coupled with the 18O-labeled internal standard approach has allowed us to show that (i) the N-domain of ACE (N-ACE), but not the C-domain, selectively cleaves the Arg-5-His-6 bond in both peptides, and that (ii) N-ACE hydrolyzes the isoAsp-7 analogue more efficiently than the non-modified one. Our results demonstrate a new endopeptidase activity of N-ACE as well as high preference of the domain to recognize and hydrolyze the isomerized Aβ species that were earlier suggested to promote AD pathogenesis. The results suggest the need for further analysis of biological effects of isomerized Aβ and its interaction with ACE in AD pathogenesis.