Treatment of human 293 cells transfected with amyloid precursor protein (APP)K595N,M596L (the "Swedish" mutation) with a specific inhibitor of the vacuolar H(+)-ATPases, bafilomycin A1 (baf A), leads to a potent inhibition of the release of the A beta peptide. This is accompanied by a selective inhibition of beta-secretase activity. Surprisingly, baf A did not inhibit the production of A beta from either wild-type APP (WT APP) or from APPv7171 (the "Hardy" mutation), expressed in the same cell type. In contrast, the robust production of A beta from a human neuroglioma-derived cell line (HS683) transfected with WT APP, or from primary human mixed brain cultures (HMBC) expressing genomic WT APP, were also effectively inhibited by baf A. The inhibition of A beta production from the HMBC was also accompanied by the inhibition of beta-s-APP release. No inhibition of alpha-s-APP release was seen in any of the cell types tested. These results indicate that intracellular acidic processes are rate-limiting for beta-secretase cleavage and A beta production from SW APP, but not WT APP, in the peripheral 293 cell line. Furthermore, such acidic processes also play a rate-limiting role in A beta release from human central nervous system-derived cells, including HMBC. Differential trafficking of the SW APP into an acidic compartment conducive to beta-secretase cleavage and A beta release could be one explanation for the increased production of A beta observed on expression of this mutation.