Aberrant production of amyloid-β peptides by processing of the β-amyloid precursor protein leads to the formation of characteristic extracellular protein deposits which are thought to be the cause of Alzheimer's disease. Therefore, inhibiting the key enzymes responsible for amyloid-β peptide generation, β- and γ-secretase may offer an opportunity to intervene with the progression of the disease. In human brain and cell culture systems a heterogeneous population of amyloid-β peptides with various truncations is detected and at present, it is unclear how they are produced. We have used a combination of surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and a specific inhibitor of γ-secretase to investigate whether the production of all amyloid-β peptide species requires the action of γ-secretase. Using this approach, we demonstrate that the production of all truncated amyloid-β peptides except those released by the action of the nonamyloidogenic α-secretase enzyme or potentially beta-site βAPP cleaving enzyme 2 depends on γ-secretase activity. This indicates that none of these peptides are generated by a separate enzyme entity and a specific inhibitor of the γ-secretase enzyme should havethe potential to block the generation of all amyloidogenicpeptides. Furthermore in the presence of γ-secretase inhibitors, the observation of increased cleavage of the membrane-bound βAPP C-terminal fragment C99 by α-secretase suggests that during its trafficking C99 encounters compartments in which α-secretase activity resides.