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Objectives. Experimental stroke studies have shown that endothelial (eNOS) and neuronal (nNOS) nitric oxide synthase isoforms exhibit opposite effects on brain injury. nNOS has been identified recently in endothelial cells, however, its functional significance is unclear. Our objective was to identify the nNOS in brain microvascular endothelial cells (BMECs) and characterize its functional role. Methods and Results. Primary BMECs from humans (hBMECs) and rats (rBMECs) were used in our studies. Immunocytochemistry identified von Willebrand factor, eNOS, and nNOS in hBMECs. Western blot analysis using antibodies targeting N-terminal domain of nNOS revealed an approximately 130 kD immunoband of a potential nNOS splice variant in hBMECs as opposed to 160 kD nNOS specific immunoband in cultured rat cortical neurons. In contrast, antibodies targeting C-terminal domain of nNOS failed to show nNOS specific immunoband. PCR experiments using the species specific primers identified the mRNA of nNOS in hBMECs. Electron Spin Resonance (ESR) spectroscopy revealed reduction of superoxide levels in hBMECs by two structurally different nNOS inhibitors, (N-ω-Propyl-L-arginine; NPA and ARL-17477), compared with vehicle (ethanol) treated cells. In contrast, treatment with eNOS inhibitor (L-N5-(1-Iminoethyl) ornithine; NIO) significantly enhanced the superoxide levels in hBMECs compared with vehicle (DMSO) treated cells. Supplementation of tetrahydrobiopterin (BH4) resulted in reduced superoxide levels in the hBMECs whereas BH4 co-treatment had no effect on the superoxide levels in the NPA or ARL-17477 treated hBMECs. NO measurements in hBMECs by ESR spectroscopy showed greatly diminished magnitude of the NO signal by the treatment with NIO, compared to vehicle (DMSO) treatment, and whereas, treatment with NPA enhanced NO signal intensity compared to vehicle (ethanol) treated cells. Conclusions. We identified a constitutively active nNOS splice variant in hBMECs that is distinct from the nNOS expressed in neurons. In addition, we found that uncoupled nNOS, significantly contributes to basal superoxide generation in hBMECs that reduces the NO bioavailability. In contrast, eNOS is the only source of NO produced by the hBMECs. We conclude that hBMECs express a unique nNOS isoform distinct from the nNOS expressed in neurons and also exhibiting effects which are distinctly opposite of eNOS.