The degradation of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) by insulin-degrading enzyme (IDE) has been investigated. As revealed by high-performance liquid chromatography, all three peptides are sequentially cleaved at a limited number of sites, the latter of which were identified by mass spectrometric analyses. The studies revealed that ANP is preferred as substrate over BNP and CNP. ANP degradation is rapidly initiated by hydrolysis at the Ser25-Phe26 bond. Three additional cleavage sites were identified in ANP after prolonged incubation with IDE; in contrast, three and two bonds were hydrolyzed in BNP and CNP, respectively. Analysis of the nine cleavage sites shows a preference for basic or hydrophobic amino acid residues on the carboxyl side of a cleaved peptide bond. In contrast to most of the peptide fragments generated by IDE activity, the initial ANP cleavage product, F-R-Y, is rapidly degraded further by cleavage of the R-Y bond. Cross-linking studies with 125I-ANP in the presence of sulfhydryl-modifying agent indicate that IDE activity is inhibited at the level of initial substrate binding whereas metal-ion chelating agents only prevent hydrolysis. On the basis of its structural and enzymatic properties, IDE exhibits striking similarity to a number of recently-described endopeptidases.