Brain accumulation and aggregation of amyloid- (A) peptides is a critical step in the pathogenesis of Alzheimer's disease (AD). Full-length A peptides (mainly A1-40 and A1-42) are produced through sequential proteolytic cleavage of the amyloid precursor protein (APP) by - and -secretases. However, studies of autopsy brain samples from AD patients have demonstrated that a large fraction of insoluble A peptides are truncated at the N-terminus, with A4-x peptides being particularly abundant. A4-x peptides are highly aggregation prone, but their origin and any proteases involved in their generation are unknown. We have identified a recognition site for the secreted metalloprotease ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) in the A peptide sequence, which facilitates A4-x peptide generation. Inducible overexpression of ADAMTS4 in HEK293 cells resulted in the secretion of A4-40 but unchanged levels of A1-x peptides. In the 5xFAD mouse model of amyloidosis, A4-x peptides were present not only in amyloid plaque cores and vessel walls, but also in white matter structures co-localized with axonal APP. In the ADAMTS4(-/-) knockout background, A4-40 levels were reduced confirming a pivotal role of ADAMTS4 in vivo. Surprisingly, in the adult murine brain, ADAMTS4 was exclusively expressed in oligodendrocytes. Cultured oligodendrocytes secreted a variety of A species, but A4-40 peptides were absent in cultures derived from ADAMTS4(-/-) mice indicating that the enzyme was essential for A4-x production in this cell type. These findings establish an enzymatic mechanism for the generation of A4-x peptides. They further identify oligodendrocytes as a source of these highly amyloidogenic A peptides.