Soluble amyloid-β (Aβ) peptide converts to structures with high β-sheet content in Alzheimer's disease (AD). Soluble Aβ is released by neurons into the brain interstitial fluid (ISF), in which it can convert into toxic aggregates. Because assessment of ISF Aβ levels may provide unique insights into Aβ metabolism and AD, an in vivo microdialysis technique was developed to measure it. Our Aβ microdialysis technique was validated ex vivo with human CSF and then in vivo in awake, freely moving mice. Using human amyloid precursor protein (APP) transgenic mice, we found that, before the onset of AD-like pathology, ISF Aβ in hippocampus and cortex correlated with levels of APP in those tissues. After the onset of Aβ deposition, significant changes in the ISF Aβ₄₀/Aβ₄₂ ratio developed without changes in Aβ_1-x. These changes differed from changes seen in tissue lysates from the same animals. By rapidly inhibiting Aβ production, we found that ISF Aβ half-life was short (∼2 hr) in young mice but was twofold longer in mice with Aβ deposits. This increase in half-life, without an increase in steady-state levels, suggests that inhibition of Aβ synthesis reveals a portion of the insoluble Aβ pool that is in dynamic equilibrium with ISF Aβ. This now measurable in vivo pool is a likely target for new diagnostic and therapeutic strategies.