It has been hypothesized that a presenilin 1 (PS1)-related enzymatic activity is responsible for proteolytic cleavage of the C-terminal intracellular protein of Notch1, in addition to its role in beta-amyloid protein (Abeta) formation from the amyloid precursor protein (APP). We developed an assay to monitor ligand-induced Notch1 proteolysis and nuclear translocation in individual cells : Treatment of full-length Notch1-enhanced green fluorescent protein-transfected Chinese hamster ovary (CHO) cells with a soluble preclustered form of the physiologic ligand Delta leads to rapid accumulation of the C terminus of Notch1 in the nucleus and to transcriptional activation of a C-promoter binding factor 1 (CBF1) reporter construct. Nuclear translocation was blocked by cotransfection with Notch's physiologic inhibitor Numb. Using this assay, we now confirm and extend the observation that PS1 is involved in Notch1 nuclear translocation and signaling in mammalian cells. We demonstrate that the D257A and the D385A PS1 mutations, which had been shown previously to block APP gamma-secretase activity, also prevent Notch1 cleavage and translocation to the nucleus but do not alter Notch1 trafficking to the cell surface. We also show that two APP gamma-secretase inhibitors block Notch1 nuclear translocation with an IC(50) similar to that reported for APP gamma-secretase. Notch1 signaling, assessed by measuring the activity of CBF1, a downstream transcription factor, was impaired but not abolished by the PS1 aspartate mutations or gamma-secretase inhibitors. Our results support the hypotheses that (a) PS1-dependent APP gamma-secretase-like enzymatic activity is critical for both APP and Notch processing and (b) the Notch1 signaling pathway remains partially activated even when Notch1 proteolytic processing and nuclear translocation are markedly inhibited. The latter is an important finding from the perspective of therapeutic treatment of Alzheimer's disease by targeting gamma-secretase processing of APP to reduce Abeta production.