Proteases that degrade the amyloid-β peptide (Aβ) are important in protecting against Alzheimer's disease (AD), and understanding these proteases is critical to understanding AD pathology. Endopeptidases sensitive to inhibition by thiorphan and phosphoramidon are especially important, because these inhibitors induce dramatic Aβ accumulation (∼30- to 50-fold) and pathological deposition in rodents. The Aβ-degrading enzyme neprilysin (NEP) is the best known target of these inhibitors. However, genetic ablation of NEP results in only modest increases (∼1.5- to 2-fold) in Aβ, indicating that other thiorphan/phosphoramidon-sensitive endopeptidases are at work. Of particular interest is the NEP homolog neprilysin 2 (NEP2), which is thiorphan/phosphoramidon-sensitive and degrades Aβ. We investigated the role of NEP2 in Aβ degradation in vivo through the use of gene knockout and transgenic mice. Mice deficient for the NEP2 gene showed significant elevations in total Aβ species in the hippocampus and brainstem/diencephalon (∼1.5-fold). Increases in Aβ accumulation were more dramatic in NEP2 knockout mice crossbred with APP transgenic mice. In NEP/NEP2 double-knockout mice, Aβ levels were marginally increased (∼1.5- to 2-fold), compared with NEP(-/-)/NEP2(+/+) controls. Treatment of these double-knockout mice with phosphoramidon resulted in elevations of Aβ, suggesting that yet other NEP-like Aβ-degrading endopeptidases are contributing to Aβ catabolism.