Sequential cleavages of the amyloid beta-protein precursor (APP) by the beta- and gamma-secretases generate the amyloid beta-protein (A beta), which plays a central role in Alzheimer's disease. Previous work provided evidence for involvement of both the secretory and endocytic pathways in A beta generation. Here, we used HeLa cells stably expressing a tetracycline-regulated dominant-negative dynamin I (dyn K44A), which selectively inhibits receptor-mediated endocytosis, and analyzed the effects on the processing of endogenous APP. Upon induction of dyn K44A, levels of mature APP rose at the cell surface, consistent with retention of APP on the plasma membrane. The alpha-secretase cleavage products of APP were increased by dyn K44A, in that alpha-APPs in medium and the C83 C-terminal stub in the membrane both rose. The beta-secretase cleavage of APP, C99, also increased modestly. The use of specific gamma-secretase inhibitors to study the accumulation of alpha- and beta-cleavage products independent of their processing by gamma-secretase confirmed that retention of APP on the plasma membrane results in increased processing by both alpha- and beta-secretases. Unexpectedly, endogenous A beta secretion was significantly increased by dyn K44A, as detected by three distinct methods: metabolic labeling, immunoprecipitation/Western blotting, and enzyme-linked immunosorbent assay. Levels of p3 (generated by sequential alpha- and gamma-cleavage) also rose. We conclude that endogenous A beta can be produced directly at the plasma membrane and that alterations in the degree of APP endocytosis may help regulate its production. Our findings are consistent with a role for the gamma-secretase complex in the processing of numerous single-transmembrane receptors at the cell surface.