-Secretase cleaves the transmembrane domain of ß-amyloid precursor protein (APP) at multiple sites referred to as -, e-, and -cleavage sites. We previously showed that DAPT, a potent dipeptide -secretase inhibitor, causes differential accumulation of longer amyloid ß-proteins (Aßs) within Chinese hamster ovary cells co-expressing ß C-terminal fragment and wild-type presenilin 1 (C99/wtPS1 cells). In this study, we used sucrose density gradient centrifugation to fractionate the membranes from C99/wtPS1 cells that had been pretreated with DAPT. We found that accumulating Aß46 localized exclusively to low-density membrane (LDM) domains. Incubating the Aß46-accumulating LDM domains at 37°C produced Aß40, Aß42, Aß43, and APP intracellular domain (AICD). The addition of L685,458 completely prevented AICD generation and resulted in a large decrease in the level of Aß46 and the concomitant appearance of Aß40 and Aß43, but not Aß42. Further addition of DAPT suppressed the production of Aß40/43 and abolished the decrease in the amount of Aß46. These data indicate that preaccumulated Aß46 is processed by -secretase to Aß40/43, but not to Aß42 in the LDM domains. The amount of newly produced Aß40 and Aß43 was roughly equivalent to the decrease in the amount of Aß46. Temporal profiles did not show a maximal concentration for Aß43, suggesting that Aß46 is processed to Aß40 and Aß43 through a nonsuccessive process.