That the zinc metalloendopeptidase insulysin (insulin-degrading enzyme IDE) is a major b-amyloid (A(beta)) peptide-degrading enzyme in vivo is shown by the higher A(beta) peptide levels in the brain of an insulysin-deficient mouse. Insulysin was shown to initially cleave A(beta)1-40and A(beta)1-42 at His13-Gln14, His14-Gln15, and Phe19-Phe20. The insulysin-dependent cleavage of A(beta) prevents both the neurotoxic effects of the peptide as well as the ability of A(beta) to deposit onto synthetic amyloid plaques. The kinetics of the reaction of insulysin with the synthetic peptide substrate Abz-G-G-F-L-R-K-H-G-Q-EDDnp displays allosteric properties indicative of a regulated enzyme. Small peptide substrates increase the activity of insulysin toward the hydrolysis of A(beta)1-40 without affecting the activity of the enzyme toward insulin. These studies indicate that insulysin is a target for drug development in which small-molecule peptide analogs can be used to increase the rate of A(beta) clearance without affecting insulin levels.