Presenilin/gamma-secretase is a membrane-associated protease that cleaves within the transmembrane region of the amyloid precursor protein (APP) to generate amyloid-beta peptide (Abeta) whose deposition in the brain is a characteristic of Alzheimer's disease (AD). Calsenilin, a calcium binding protein that has been shown to interact with the C-termini of both presenilin 1 (PS1) and presenilin 2 (PS2), appears to play a role in transcriptional regulation and apoptosis and to bind to A-type voltage-gated potassium channels. Here, we report that overexpression of calsenilin enhanced gamma-secretase activity in cells. The effect of calsenilin on the gamma-cleavage of substrates was blocked by the selective gamma-secretase inhibitor L-685,458. We also employed a cellular gamma-cleavage GFP-reporter assay to demonstrate the effect of calsenilin on gamma-secretase activity. To establish a direct role for calsenilin in regulating gamma-secretase activity, we incubated purified calsenilin with isolated membrane fractions and found increased Abeta production in a cell free system. These data suggest that calsenilin may be one of the regulatory factors for gamma-secretase.