Sepsis-associated encephalopathy is an early manifestation of sepsis, resulting in a diffuse dysfunction of the brain. Recently, nitric oxide (NO) has been proposed to be one of the key molecules involved in the modulation of inflammatory responses in the brain. The aim of this study was to assess the role of NO in cerebrovascular endothelial cell activation/dysfunction during the early onsets of sepsis. To this end we employed an in vitro model of sepsis in which cultured mouse cerebrovascular endothelial cells (MCVEC) were challenged with blood plasma (20 % v/v) obtained from sham or septic (feces-induced peritonitis, FIP-6 hrs) mice. Exposing MCVEC to FIP-plasma for 1 hr resulted in increased production of ROS and NO as assessed by intracellular oxidation of oxidant-sensitive fluorochrome, DHR 123, and nitrosation of NO-specific probe, DAF-FM, respectively. The latter events were accompanied by dissociation of tight junction protein, occludin, from MCVEC cytoskeletal framework and a subsequent increase in FITC-Dextran (MW-3kDa) flux across MCVEC grown on the permeable cell culture supports, whereas Evans blue-BSA (MW-65kDa) or FITC-Dextran (MW-10kDa) flux were not affected. FIP-plasma induced oxidant stress, occludin rearrangement and MCVEC permeability were effectively attenuated by anti-oxidant, PDTC (0.5mM) or interfering with NOS activity (L-NAME, 0.1 mM, or eNOS^-/- MCVEC). However, treatment of MCVEC with PDTC failed to interfere with NO production, suggesting that septic plasma-induced oxidant stress in MCVEC is primarily NO-dependent event. Taken together these data indicate that during early sepsis eNOS-derived NO exhibits pro-inflammatory characteristics and contributes to the activation and dysfunction of cerebrovascular endothelial cells.