Although tau is a cytoplasmic protein, it is also found in brain extracellular fluids, e.g., CSF. Recent findings suggest that aggregated tau can be transferred between cells and extracellular tau aggregates might mediate spread of tau pathology. Despite these data, details of whether tau is normally released into the brain interstitial fluid (ISF), its concentration in ISF in relation to CSF, and whether ISF tau is influenced by its aggregation are unknown. To address these issues, we developed a microdialysis technique to analyze monomeric ISF tau levels within the hippocampus of awake, freely moving mice. We detected tau in ISF of wild-type mice, suggesting that tau isreleased in the absence of neurodegeneration. ISF tau was significantly higher than CSF tau and their concentrations were not significantly correlated.UsingP301S human tau transgenicmice (P301Stgmice),we found that ISFtauisfivefoldhigherthan endogenousmurinetau, consistent withits elevated levelsofexpression. However, following the onsetoftauaggregation, monomericISF tau decreased markedly. Biochemical analysis demonstrated that soluble tau in brain homogenates decreased along with the deposition of insoluble tau. Tau fibrils injected into the hippocampus decreased ISF tau, suggesting that extracellular tau is in equilibrium with extracellular or intracel-lular tau aggregates. This technique should facilitate further studies of tau secretion, spread of tau pathology, the effects of different disease states on ISF tau, and the efficacy of experimental treatments.