Amyloid beta peptide (A beta) is released into the media of a variety of cells in culture during normal metabolism. The discovery of several missense mutations within or flanking the A beta region of the beta amyloid precursor protein (beta APP) in familial Alzheimer's disease provides strong evidence for a role of altered processing of beta APP in the pathogenesis of this disorder. The cellular mechanisms that regulate the relative utilization of the secretory pathway, which causes beta APP to be cleaved within the A beta domain, and the alternative proteolytic pathway, which produces intact A beta, are unknown. It is hypothesized that a number of neurodegenerative diseases, including Alzheimer's disease, are characterized by abnormal calcium metabolism. We investigated the effect of disordered calcium homeostasis on A beta production in human kidney 293 cells transfected with beta APP cDNA. A beta immunoprecipitated from the conditioned media of cells was compared to immunoprecipitated full-length and secreted forms of beta APP in both metabolic labeling and pulse-chase labeling paradigms. The calcium ionophore A23187 consistently increased the production of A beta approximately 3-fold. This effect was dependent on the presence of extracellular calcium in intact cells. Caffeine also increased A beta production, possibly through release of calcium from intracellular stores. The increase in A beta was cAMP-independent, and it was not mediated by a protein kinase C-dependent pathway, as treatment with phorbol esters decreased A beta levels. The effects of the ionophore on beta APP maturation and phosphorylation were also established. We conclude that elevation of intracellular calcium levels has an important effect on beta APP maturation and proteolytic processing and substantially enhances the production and release of the amyloidogenic A beta peptide.