Intramembrane proteolysis of transmembrane substrates by the presenilin–-secretase complex is preceded and regulated by shedding of the substrate’s ectodomain by α- or β-secretase. We asked whether β- and -secretases interact to mediate efficient sequential processing of APP, generating the amyloid β (Aβ) peptides that initiate Alzheimer’s disease. We describe a hitherto unrecognized multiprotease complex containing active β- and -secretases. BACE1 coimmunoprecipitated and cofractionated with -secretase in cultured cells and in mouse and human brain. An endogenous high molecular weight (HMW) complex (~5 MD) containing β- and -secretases and holo-APP was catalytically active in vitro and generated a full array of Aβ peptides, with physiological Aβ42/40 ratios. The isolated complex responded properly to -secretase modulators. Alzheimer’s-causing mutations in presenilin altered the Aβ42/40 peptide ratio generated by the HMW β/-secretase complex indistinguishably from that observed in whole cells. Thus, Aβ is generated from holo-APP by a BACE1–-secretase complex that provides sequential, efficient RIP processing of full-length substrates to final products.