Proteolytic processing of the amyloid precursor protein (APP) generates amyloid β (Aβ) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by β-secretase at the amino terminus of the Aβ peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of β-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by γ-secretase(s) leads to the formation of Aβ. The pathogenic mutation K670M671 → N670L671 at the β-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased β-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the β-secretase cleavage site, and find it to be the predominant β-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for β-secretase. Cloning and expression of the enzyme reveals that human brain β-secretase is a new membrane-bound aspartic proteinase.