Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-β protein, which is central to the pathogenesis of Alzheimer's disease. It has been demonstrated that PSI regulates the γ-secretase proteolysis of the amyloid precursor protein (APP) C-terminal fragment (APP- C100), which is the final step in amyloid-β protein production. The mechanism and detailed pathway of this PSI activity has yet to be fully resolved, but it may be due to a presenilin-controlled trafficking of the APP fragment or possibly an inherent PSI proteolytic activity. We have investigated the possibility of a direct interaction of PSI and the APP-C100 within the high molecular mass presenilin complex. However, the APP-C100 is rapidly degraded, and if it forms, then any PS1 APP complex is likely to be very transitory. To circumvent this problem, we have utilized the protease inhibitor N-acetyl-leucyl-norleucinal (LLnL) and the lysosomotropic agent NH4Cl, which inhibits the turnover of the APP-C100. Under these conditions, levels of the fragment increased appreciably, and as shown by glycerol gradient analysis, the APP-C100 shifted to a higher molecular mass complex that overlapped with PSI. Immunoprecipitation studies demonstrated that a significant population of the APP-C100 co-precipitated with PS1. These findings suggest that PSI may mediate the shuttling of APP fragments and/or facilitate their presentation for γ-secretase cleavage through a direct interaction.