The amyloid precursor protein (APP) undergoes abnormal metabolism in Alzheimer's disease, resulting in the accumulation of βA4 amyloid in the brain. Normal APP metabolism includes the release of a truncated form (sAPP) which has been cleaved at the a-secretase site within the βA4 amyloidogenic domain. However, intact forms of βA4 protein may also be generated by the β- and γ-secretases. Soluble forms of βA4 have been detected in various cell lines and in cerebrospinal fluid. Previous studies of protein kinase C activation have suggested a reciprocal relationship between sAPP secretion and βA4 production and release. We find that phorbol ester activation of protein kinase C in untransfected SHSY5Y neuroblastoma cells increases the release of sAPP without affecting βA4 secretion. We provide further evidence for intracellular βA4 production. Treatment of SY5Y cells with the protease inhibitor phosphoramidon results in a 2-fold increase in βA4 secretion and an increase in the amount of βA4 recovered from cell lysates, yet it does not affect sAPP secretion. The protease inhibitors thiorphan and N-[(RS)-2-carboxy-3-phenylpropanoyl]-L-leucine had no effect on βA4 or sAPP secretion. The lysosomotropic agents chloroquine and NH4Cl decreased βA4 secretion, providing additional evidence for the involvement of intracellular acidic compartments in the production of βA4. Our results therefore demonstrate a double dissociation between the secretion of sAPP and βA4 in the SH-SY5Y cell line. The effect of phosphoramidon supports previous studies which show that metalloproteases are involved in the biogenesis of βA4.