Mutations in the presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease by altering γ-secretase cleavage of the amyloid precursor protein, the last step in the generation of Aβ peptide. Ablation of presenilin (PS) genes, or mutation of two critical aspartates, abolishes γ-secretase cleavage, suggesting that PS may be the γ-secretases. Independently, inhibition experiments indicate that γ-secretase is an aspartyl protease. To characterize the putative γ-secretase activity associated with presenilins, lysates from human neuroblastoma SH-SY5Y and human brain homogenates were incubated with biotin derivatives of pepstatin, followed by immunoprecipitation of PS and associated proteins, and biotin detection by Western blotting. Precipitation with PS1 antibodies, directed to either N-terminal or loop regions, yielded the same 43 kDa band, of apparent molecular mass consistent with that of full-length PS1, although it may represent an aspartyl protease complexed with PS1. Incubation of cell lysates with pepstatin−biotin, followed by streptavidin precipitation and PS1 Western blotting, revealed PS1 fragments and full-length protein, indicating that pepstatin−biotin bound to both cleaved and uncleaved PS1. Binding could be competed by γ-secretase inhibitor L-685,458 and could not be achieved with a PS1 mutant lacking the two transmembrane aspartates. Pepstatin−biotin was also shown to bind to PS2. PS1 was specifically absorbed to pepstatin−agarose, with an optimal pH of 6. Binding of pepstatin−biotin to PS1 from lymphocytes of a heterozygous carrier of pathologic exon 9 deletion was markedly decreased as compared to control lymphocytes, suggesting that this PS1 mutation altered the pepstatin binding site.