Cerebral microvascular dysfunction has been implicated in the brain injury following stroke, however, the underlying mechanisms are unclear. Nitric oxide synthase (NOS) has endothelial (eNOS) and neuronal (nNOS) isoforms that were named after the locations where they were first identified. Our preliminary studies, for the first time, identified nNOS in freshly isolated rat brain microvessels and brain microvascular endothelial cells (BMECs) from rat, mouse, and humans utilizing PCR and immunoblot techniques. We found that endothelial nNOS is structurally and functionally distinct from eNOS and the nNOS expressed in the neurons. Therefore, we named the endothelial nNOS as enNOS. Our preliminary studies revealed that inhibition of eNOS in BMECs or nNOS in neurons increased the levels of superoxide and decreased NO levels. Furthermore, eNOS inhibition results in diminished mitochondrial reserve respiratory capacity. Similarly, inhibition of nNOS in neurons increased superoxide levels and decreased NO levels. In contrast, enNOS inhibition led to diminished superoxide levels, increased NO levels, and enhanced mitochondrial reserve respiratory capacity. Thus, unlike nNOS of neuronal origin and eNOS, enNOS exists in the uncoupled state. Preliminary studies also showed that enNOS significantly contributes to baseline as well as angiotensin II- induced superoxide levels in BMECs that is comparable to but independent of NADPH oxidase. Finally, inhibition of all NOS isoforms during oxygen-glucose deprivation and reoxygenation (OGD-R) decreased superoxide generation from cytosol and mitochondrial sources resulting in increased survival of BMECs which indicates the physiological significance of enNOS in BMECs. Experimental stroke-induced brain damage is greater in eNOS but diminished in nNOS knockouts, however, the exact mechanisms underlying the nNOS inhibition afforded neuroprotection have never been examined. We hypothesize that enNOS is functionally distinct from eNOS and nNOS of neuronal origin. We further hypothesize that enNOS is the primary mediator of OGD-R injury to BMECs and is an important modulator of post-ischemic BBB disruption. Aim 1 will demonstrate that enNOS is functionally distinct from eNOS and nNOS in generating superoxide versus NO and in modulating mitochondrial function after OGD-R in cultured BMECs and neurons. Aim 2 will determine the functional significance of enNOS and eNOS on post-OGD-R viability and structural integrity of BMECs. Aim 3 will determine the differential role of enNOS and eNOS on the post-ischemic BBB integrity and microvascular dysfunction. The proposed studies will fundamentally advance our mechanistic understanding of NOS, the single most important regulator of neurovascular unit, and will provide breakthrough findings to target enNOS for treating microvascular dysfunction in stroke.