Amyloidogenic processing of the β-amyloid precursor protein (APP) has been implicated in the pathology of Alzheimer's disease. Because it has been suggested that catabolic processing of the APP holoprotein occurs in acidic intracellular compartments, we studied the effects of the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) and the H+-ATPase inhibitor bafilomycin A1 on APP catabolism in human embryonic kidney 293 cells expressing either wild-type or "Swedish" mutant APP. Unlike bafilomycin A1, which inhibits β-amyloid production in cells expressing mutant but not wild-type APP, FCCP inhibited β-amyloid production in both cell types. Moreover, the effects of FCCP were independent of alterations in total cellular APP levels or APP maturation, and the concentrations used did not alter either cellular ATP levels or cell viability. Bafilomycin A1, which had no effect on β-amyloid production in wild-type cells, inhibited endocytosis of fluorescent transferrin, whereas concentrations of FCCP that inhibited β-amyloid production in these cells had no effect on endosomal function. Thus, in wild-type-expressing cells it appears that the β-amyloid peptide is not produced in the classically defined endosome. Although bafilomycin A1 decreased β-amyloid release from cells expressing mutant APP but not wild-type APP, it altered lysosomal function in both cell types, suggesting that in normal cells β-amyloid is not produced in the lysosome. Although inhibition of β-amyloid production by bafilomycin A1 in mutant cells may occur via changes in endosomal/lysosomal pH, our data suggest that FCCP inhibits wild-type β-amyloid production by acting on a bafilomycin A1-insensitive acidic compartment that is distinct from either the endosome or the lysosome.