Since the first report showing that Alzheimer disease (AD) might be caused by mutations in the amyloid precursor protein gene (APP), 20 different missense mutations have been reported. The majority of early-onset AD mutations alter processing of APP increasing relative levels of A beta 42 peptide, either by increasing A beta 42 or decreasing A beta 40 peptide levels or both. In a diagnostic setting using direct sequence analysis, we identified in one patient with familial early-onset AD a novel mutation in APP (c.2172G > C), predicting a K724N substitution in the intracytosolic fragment. The mutation is located downstream of the epsilon-cleavage site of APP and is the furthermost C-terminal mutation reported to date. In vitro expression of APP K724N cDNA showed an increase in A beta 42 and a decrease in A beta 40 levels resulting in a near three-fold increase of the A beta 42/A beta 40 ratio. Further, in vivo amyloid positron emission tomography (PET) imaging revealed significantly increased cortical amyloid deposits, supporting that in human this novel APP mutation is likely causing disease.