Presenilin-dependent intramembranous proteolysis mediates the dual cleavage of the Notch-1 protein (S4 and S3) as well as the beta amyloid precursor protein (betaAPP) (gamma40 and epsilon-site). betaAPP has a valine residue just before the gamma40 (amyloid beta [Abeta] numbering) site and after the epsilon-site. Both gamma40 and epsilon have multiple cleavage sites, and the varieties of gamma40 cleavage are associated with Alzheimer's disease (AD). These lines of evidence suggest that valine plays a role in the intramembranous proteolysis. S4 cleavage in the middle of the Notch-1 transmembrane domain (TMD) corresponds to the gamma40 cleavage of betaAPP. The cleavage site is in the center of four sequential alanine residues between Ala1731 and Ala1732, neither of which has a valine residue. To investigate the effects of valine on presenilin-dependent intramembranous proteolysis, we replaced the transmembrane domain residue of Notch-1 with valine and analyzed the efficiency and precision at S4 and S3. We observed that all valine-mutated Notch-1 proteins have a dominant cleavage site (S4) between Ala1731 and Ala1732 with some variations of cleavage precision, suggesting that valine is not indispensable for determining the cleavage site of the Notch-1 transmembrane domain, but affects the efficiency and precision at S4 cleavage of the Notch-1 transmembrane domain.