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The first goal of the present study was to determine the effect of tumor necrosis factor-α (TNF-α) on the permeability of the blood–brain barrier in vivo. The second goal of this study was to investigate cellular pathways responsible for changes in permeability of the blood–brain barrier in response to TNF-α. We examined the pial microcirculation in rats using intravital fluorescence microscopy. Permeability of the blood–brain barrier was quantitated by calculating the clearance of fluorescent-labeled dextran (mol. wt=10,000; FITC–dextran-10K) during superfusion with vehicle, tumor necrosis factor (TNF-α; 10 ng/ml), TNF-α in the presence of an inhibitor of soluble guanylate cyclase (ODQ; 1.0 μM), and TNF-α in the presence of an inhibitor of protein tyrosine kinase (genistein; 10 μM). During superfusion with vehicle, clearance of FITC–dextran-10K from pial vessels remained relatively constant during the experimental …