We investigated the processing pathway of the amyloid precursor protein (APP) to the secretion of beta A4 under the treatment of ammonium chloride (NH4Cl), bafilomycin A1 (bafA1), or chloroquine, all three agents thought to raise the pH in acidic compartments. HEK-293 cells expressing wild-type APP (APPwt) and APP carrying the Swedish double mutation (APPswe) were affected in a different manner: while cells expressing APPswe decreased the secretion of beta A4 after treatment with bafA1 and NH4Cl, cells expressing APPwt compensated the drug-induced decrease in beta A4 by an increased generation of alternative beta A4-related peptides. Within cells APP accumulated, while the formation of a C-terminal fragment of APP generated by beta-secretase was completely inhibited. Thus, BafA1 and NH4Cl reduced the secretion of beta A4 by inhibiting beta-secretase. Treatment with chloroquine did not alter beta A4 secretion but, strikingly, resulted in an accumulation of intracellular beta A4. The effect of reduced APP endocytosis was studied by expressing APP molecules lacking the cytoplasmic domain (APPwt.delta. APPswe.delta). Truncation of APP reduced beta A4 secretion from APPwt but not from APPswe. BafA1 and NH4Cl treatment inhibited the formation of beta A4 in cells expressing APPswe.delta but not APPwt.delta. With these constructs, chloroquine had no effect and no accumulation of intracellular beta A4 was observed. Since alkalizing agents still affected endocytosis-deficient APP containing the Swedish double mutation, we suggest that the formation of beta A4 from this mutated APP takes place mainly in an acidic compartment along the constitutive secretory pathway. Much in contrast to this, beta A4 generation from APPwt appears to occur also in the endosomal/lysosomal compartment.