Production of amyloid β peptides (Aβ), followed by their deposition in the brain as amyloid plaques, contributes to the hallmark pathology of Alzheimer disease. The enzymes responsible for production of Aβ, BACE1 and γ-secretase, are therapeutic targets for treatment of Alzheimer disease. Two presenilin (PS) homologues, referred to as PS1 and PS2, comprise the catalytic core of γ-secretase. In comparing presenilin selectivity of several classes of γ-secretase inhibitors, we observed that sulfonamides in general tend to be more selective for inhibition of PS1-comprising γ-secretase, as exemplified by ELN318463 and BMS299897. We employed a combination of chimeric constructs and point mutants to identify structural determinants for PS1-selective inhibition by ELN318463. Our studies identified amino acid residues Leu172, Thr281, and Leu282 in PS1 as necessary for PS1-selective inhibition by ELN318463. These residues also contributed in part to the PS1-selective inhibition by BMS299897. Alanine scanning mutagenesis of areas flanking Leu172, Thr281, and Leu282 identified additional amino acids that affect inhibitor potency of not only these sulfonamides but also nonsulfonamide inhibitors, without affecting Aβ production and presenilin endoproteolysis. Interestingly, many of these same residues have been identified previously to be important for γ-secretase function. These findings implicate TM3 and a second region near the carboxyl terminus of PS1 aminoterminal fragment in mediating the activity of γ-secretase inhibitors. Our observations demonstrate that PS-selective inhibitors of γ-secretase are feasible, and such inhibitors may allow differential inhibition of Aβ peptide production and Notch signaling.