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The blood–brain barrier (BBB) is critical to the health of the central nervous system. The BBB is formed primarily by the presence of tight junctions (TJ) between cerebral microvessel endothelial cells. In light of the known effects of nicotine on endothelial cell biology, the specific effects of nicotine on the in vivo BBB were examined. Using in situ brain perfusion, it was found that continuous administration of nicotine (4.5 mg free base·kg?1·day?1) for 1 and 7 days led to increased permeability of the BBB to [14C]-sucrose without significant changes in its initial volume of distribution. The expression and distribution of the TJ-associated proteins actin, occludin, claudin-1, -3, and -5, and ZO-1 and -2 were analyzed by Western blot and immunofluorescence microscopy. Though no changes in total protein expression were observed, nicotine treatment was associated with altered cellular distribution of ZO-1 and diminished junctional immunoreactivity of claudin-3. It is proposed that nicotine leads to changes in BBB permeability via the modulation of TJ proteins.