β-Amyloid peptide accumulates in the brain of patients affected by sporadic or familial forms of Alzheimer's disease. It derives from the proteolytic attacks of the β-amyloid precursor protein (βAPP) by β- and γ-secretase activities. An additional η cleavage taking place a few residues C-terminal to the γ-site has been reported, leading to the formation of an intracellular fragment referred to as APP intracellular domain C50. This η cleavage received particular attention because it resembles the S3 Notch cleavage generating Notch intracellular domain. Indeed, APP intracellular domain, like its Notch counterpart, appears to mediate important physiological functions. γ and η cleavages on βAPP appear spatio-temporally linked but pharmacologically distinct and discriminable by mutagenesis approaches. As these cleavages could be seen as either deleterious (γ-site) or beneficial (η-site), it appears of most interest to set up models aimed at studying these activities separately, particularly to design specific and bioavailable inhibitors. On the other hand, it is important to respect the topology of the substrates in order to examine physiologically relevant cleavages. Here we describe the obtention of cells overexpressing APPη, the η-secretase-derived N-terminal fragment of βAPP. Interestingly, this N-terminal fragment of βAPP was shown by biochemical and immunohistochemical approaches to behave as a genuine membrane-bound protein. APPη undergoes constitutive and protein kinase C-regulated α-secretase cleavages. Furthermore, APPη is targeted by the β-secretase β-site APP-cleaving enzyme and is subsequently cleaved by γ-secretase. The resulting β-amyloid peptide production is fully prevented by various γ-secretase inhibitors. Altogether, our study shows that APPη is a relevant βAPP derivative to study γ-secretase activities and to design specific inhibitors without facing any rate-limiting effect of η-secretase-derived cleavage.