The abnormal accumulation of β-amyloid (Aβ) in the brain is an early and invariant feature in Alzheimer's disease (AD) and is believed to play a pivotal role in the etiology and pathogenesis of the disease. As such, a major focus of AD research has been the elucidation of the mechanisms responsible for the generation of Aβ. As with any peptide, however, the degree of Aβ accumulation is dependent not only on its production but also on its removal. In cell-based and in vitro models we have previously characterized endothelin-converting enzyme-1 (ECE-1) as an Aβ-degrading enzyme that appears to act intracellularly, thus limiting the amount of Aβ available for secretion. To determine the physiological significance of this activity, we analyzed Aβ levels in the brains of mice deficient for ECE-1 and a closely related enzyme, ECE-2. Significant increases in the levels of both Aβ40 and Aβ42 were found in the brains of these animals when compared with age-matched littermate controls. The increase in Aβ levels in the ECE-deficient mice provides the first direct evidence for a physiological role for both ECE-1 and ECE-2 in limiting Aβ accumulation in the brain and also provides further insight into the factors involved in Aβ clearance in vivo.