Human somatic angiotensin-converting enzyme (ACE) is a membrane-bound dipeptidyl carboxypeptidase that contains two extracellular domains (N and C). Although highly homologous, they exhibit different substrate and inhibition profiles. The phosphinic inhibitors RXPA380 and RXP407 are highly selective for the C- and N-domains, respectively. A number of residues, implicated by structural data, are likely to contribute to this selectivity. However, the extent to which these different interactions are responsible for domain selectivity is unclear. In this study, a series of C- and N-domain mutants containing conversions to corresponding domain residues were used to scrutinize the contribution of these residues to selective inhibitor binding. Enzyme kinetic analyses of the purified mutants indicated that the RXPA380 C-selectivity is particularly reliant on the interaction between the P2 substituent and Phe 391 (testis ACE numbering). Moreover, a C-domain mutant in which Phe 391 has been changed to a Tyr residue, in addition to containing an N-domain S2' pocket (S2'F/Y), displayed the greatest shift toward a more N-domain-like Ki. None of the single mutations within the N-domain caused a large shift in RXP407's affinity for these enzymes. However, the double mutant containing the Tyr 369 to Phe change as well as Arg 381 to Glu displayed a 100-fold decrease in binding affinity, confirming that the S2 pocket plays a major role in RXP407 selectivity. Taken together, these data advance our understanding regarding the molecular basis for the remarkable ACE domain selectivity exhibited by these inhibitors.