Presenilins 1 and 2 (PS1/PS2) have been suggested to be γ-secretases responsible for the proteolytic cleavage of amyloid precursor protein (APP) to form amyloid-β (Aβ), a protein implicated in the development of Alzheimer's disease. Here we examined whether these presenilins are required for the generation of multiple Aβ species by analyzing the production of several forms of secreted and intracellular Aβ in mouse cells lacking PS1, PS2 or both proteins. Although most Aβ species were abolished in PS1/PS2^−/− cells, the production of intracellular Aβ42 generated in the endoplasmic reticulum/intermediate compartment was unaffected by the absence of these proteins, either singly or in combination. These results indicate that production of this pool of Aβ occurs independently of PS1/PS2, and therefore, another γ-secretase activity must be responsible for cleavage of APP within the early secretory compartments.