Nicastrin (NCT) is a type I integral membrane protein that is one of the four essential components of the ␥-secretase complex, a protein assembly that catalyzes the intramembranous cleavage of the amyloid precursor protein and Notch. Other ␥-secretase components include presenilin-1 (PS1), APH-1, and PEN-2, all of which span the membrane multiple times. The mechanism by which NCT associates with the ␥-secretase complex and regulates its activity is unclear. To avoid the misfolding phenotype often associated with introducing deletions or mutations into heavily glycosylated and disulfidebonded proteins such as NCT, we produced chimeras between human (hNCT) and Caenorhabditis elegans NCT (ceNCT). Although ceNCT did not associate with human ␥-secretase components, all of the ceNCT/hNCT chimeras interacted with ␥-secretase components from human, C. elegans, or both, indicating that they folded correctly. A region at the C-terminal end of hNCT, encompassing the last 50 residues of its ectodomain, the transmembrane domain, and the cytoplasmic domain was important for mediating interactions with human PS1, APH-1, and PEN-2. This finding is consistent with the fact that the bulk of the ␥-secretase complex proteins resides within the membrane, with relatively small extramembranous domains. Finally, hNCT associated with hAPH-1 in the absence of PS, consistent with NCT and APH-1 forming a subcomplex prior to association with PS1 and PEN-2 and indicating that the interactions between NCT with PS1 may be indirect or stabilized by the presence of APH-1.