Over the last few years, we have identified an orally active, novel neuroprotective and cognition-enhancing molecule, the flavonoid fisetin. Fisetin not only has direct antioxidant activity but it can also increase the intracellular levels of glutathione, the major intracellular antioxidant. Fisetin can also activate key neurotrophic factor signaling pathways. In addition, it has anti-inflammatory activity against microglia and astrocytes and inhibits the activity of lipoxygenases, thereby reducing the production of pro-inflammatory eicosanoids and their by-products. However, key questions about its targets and brain penetration remain. In this study, we used label-free two-photon microscopy of intrinsic fisetin fluorescence to examine the localization of fisetin in living nerve cells and the brains of living mice. In cells, fisetin but not structurally related flavonols with different numbers of hydroxyl groups, localized to the nucleoli suggesting that key targets of fisetin may reside in this organelle. In the mouse brain, following intraperitoneal injection and oral administration, fisetin rapidly distributed to the blood vessels of the brain followed by a slower dispersion into the brain parenchyma. Thus, these results provide further support for the effects of fisetin on brain function. In addition, they suggest that label-free two-photon microscopy may prove useful for studying the intracellular and tissue distribution of other intrinsically-fluorescent flavonoids.