Presenilin (PS), the catalytic subunit of γ-secretase, is present in two homologous variants (PS1 and PS2) in human cells. The analysis of PS1, PS2, and chimeric PS1/2 variants enabled the identification of PS domains that are responsible for differences in C99 cleavage between the homologs. We found that PS cleavage efficiency is determined by transmembrane domain (TMD)6, TMD8, TMD9, and the combination of TMD3 and TMD4. Furthermore, TMD3 was identified as a major modulator of initial cleavage.