The small-vessel disorder cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) arises from mutations in the human gene encoding NOTCH3 and results in vascular smooth muscle cell degeneration, stroke, and dementia. However, the structural changes in NOTCH3 involved in CADASIL etiology are unclear. Here, we discovered site-specific fragmentation of NOTCH3 protein in pathologically affected vessels of human CADASIL-affected brains. EM-based experiments to pinpoint NOTCH3 localization in these brains indicated accumulation of NOTCH3 fragmentation products in the basement membrane, collagen fibers, and granular osmiophilic material (GOM) within the cerebrovasculature. Using antibodies generated against a disease-linked neo-epitope found in degenerating vascular media of CADASIL brains, we mapped the site of fragmentation to the NOTCH3 N-terminus at the peptide bond joining Asp-80 and Pro-81. Cleavage at this site was predicted to separate the first epidermal growth factor (EGF)-like domain from the remainder of the protein. We found that the cleavage product from this fragmentation event is released into the conditioned media of cells expressing recombinant NOTCH3 fragments. Mutagenesis of Pro-81 abolished the fragmentation, and low pH and reducing conditions enhanced NOTCH3 proteolysis. Furthermore, substitution of multiple cysteine residues of the NOTCH3 N-terminus activated proteolytic release of the first EGF-like repeat, suggesting that elimination of multiple disulfide bonds in NOTCH3 accelerates its fragmentation. These characteristics link the signature molecular genetic alterations present in individuals with CADASIL to a post-translational protein alteration in degenerating brain arteries. The cellular consequences of these pathological NOTCH3 fragments are an important area for future investigation.,Published under license by The American Society for Biochemistry and Molecular Biology, Inc.