Aggregation of amyloid-β (Aβ) peptide into soluble and insoluble forms within the brain extracellular space is central to the pathogenesis of Alzheimer's disease. Full-length amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce Aβ. Aβ is subsequently released into the brain interstitial fluid (ISF). We hypothesized that synaptic transmission results in more APP endocytosis, thereby increasing Aβ generation and release into the ISF. We found that inhibition of clathrin-mediated endocytosis immediately lowers ISF Aβ levels in vivo. Two distinct methods that increased synaptic transmission resulted in an elevation of ISF Aβ levels. Inhibition of endocytosis, however, prevented the activity-dependent increase in Aβ. We estimate that ∼70% of ISF Aβ arises from endocytosis-associated mechanisms, with the vast majority of this pool also dependent on synaptic activity. These findings have implications for AD pathogenesis and may provide insights into therapeutic intervention.