Two β-secretases, BACE1 and BACE2, are involved in generation of Alzheimer's disease Aβ peptides 1, 2, 3. We report that secretion of Aβ peptides (Aβ1–40/42 and Aβ11–40/42) is abolished in cultures of BACE1-deficient embryonic cortical neurons, and that whereas both human and murine BACE1 can cleave either human or murine β-amyloid precursor protein (APP) at the+ 1 site of Aβ, cleavage at the+ 11 site is species specific. We establish that BACE1 is the principal neuronal protease required to cleave APP at+ 1 and+ 11 sites that generate N-termini of Aβ.