Angiotensin I-converting enzyme (ACE; kininase II) contains two very similar domains (the NH2- and COOH-terminal domains (N and C domains, respectively)), each bearing an active site. These active sites hydrolyze the same peptides, but do not have the same catalytic properties and substrate specificities. In an attempt to develop domain-specific immunological probes, two series of monoclonal antibodies (mAbs), 19 clones in all, were produced and tested against human ACE. These mAbs recognized at least nine different epitopes within three antigenic regions of the ACE molecule. Testing on wild-type recombinant ACE and several mutants with only one intact domain showed that these epitopes were all located in the N domain. None of the mAbs recognized the C domain. This particular specificity and analysis of results obtained with several polyclonal antibodies to human ACE suggest that ACE immunogenicity is determined mainly by the N domain. Two mAbs (3A5 and i2H5) recognizing epitopes from different antigenic regions of ACE inhibited the enzymatic activity of the N (but not of the C) domain. mAb 3A5 had the same inhibitory potency toward hippuryl-His-Leu, benzyloxycarbonyl-Phe-His-Leu, and angiotensin I hydrolysis, with 50% inhibition achieved at a mAb/ACE molar ratio of 6. mAb i2H5 was roughly three times more effective than mAb 3A5 inhibiting the hydrolysis of benzyloxycarbonyl-Phe-His-Leu and the natural substrates angiotensin I and bradykinin (50% inhibition at a molar ratio of 1-2), but was less effective in inhibiting hippuryl-His-Leu cleavage (50% inhibition at a molar ratio of 22-25), indicating that this substrate interacts with a specific subsite. mAb i2H5 almost completely inhibited the hydrolysis of the luteinizing hormone-releasing hormone by the isolated N domain. Both the primary carboxyl- and amino-terminal cleavages of this peptide were suppressed. This antibody suppressed the primary amino-terminal cleavage of the luteinizing hormone-releasing hormone by wild-type ACE by > 90%, indicating that this particular ACE function is mediated mainly by the N domain active site. These data provide evidence for structural differences between the two homologous domains of ACE despite their high degree of sequence homology and show that monoclonal antibodies are able to distinguish between the two active sites in ACE.