Abstract Background Several familial Alzheimer disease (FAD) mutations within the transmembrane region of the amyloid precursor protein (APP) increase the Aβ42/40 ratio without increasing total Aβ production. In the present study, we analyzed the impact of FAD mutations and γ-secretase modulators (GSMs) that alter the Aβ42/40 ratio on APP C-terminus (CT) positioning relative to the membrane, reasoning that changes in the alignment of the APP intramembranous domain and presenilin 1 (PS1) may impact the PS1/γ-secretase cleavage site on APP. Results By using a Förster resonance energy transfer (FRET)-based technique, fluorescent lifetime imaging microscopy (FLIM), we show that Aβ42/40 ratio-modulating factors which target either APP substrate or PS1/γ-secretase affect proximity of the APP-CT to the membrane and change PS1 conformation. Conclusions Thus, we propose that there is a reciprocal relationship between APP-CT positioning relative to the membrane and PS1 conformation, suggesting that factors that modulate either APP positioning in the membrane or PS1 conformation could be exploited therapeutically.