Somatic angiotensin I-converting enzyme (ACE)has two homologous active sites (N and C domains) that show differences in various biochemical properties.In a previous study, we described the use of positionals canning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides to define the ACE C-domain versus N-domain substrate specificity and developed selective substrates for the C-domain(Bersanetti et al., 2004). In the present work, we used the results from the PS-SC libraries to define the N-domain preferences and designed selective substrates for this domain. The peptide Abz-GDDVAK(Dnp)-OH presented the most favorable residues for N-domain selectivity in the P 3 to P 1 ′ positions. The fluorogenic analog Abz-DVAK(Dnp)-OH (Abz = ortho -aminobenzoic acid; Dnp = 2,4-dinitrophenyl)showed the highest selectivity for ACE N-domain( k cat /K m = 1.76 μ m -1 · s -1) . Systematic reduction of the peptide length resulted in a tripeptide that was preferentially hydrolyzed by the C-domain. The binding of Abz-DVAK(Dnp)-OH to the active site of ACE N-domain was examined using a combination of conformational analysis and molecular docking. Our results indicated that the binding energies for the N-domain-substrate complexes were lower than those for the C-domain-substrate, suggesting that the former complexes are more stable.