The etiology of stroke, a major form of vaso-occlusion in hemoglobin SS(HgbSS) disease, remains unclear, but may involve red blood cell (RBC)adhesion and altered vascular tone regulation. We hypothesized that nitric oxide (NO) participates in maintaining cerebral blood flow in HgbSS disease and its inhibition would result in vaso-occlusion in an in vivo cerebral microcirculation model. Laser Doppler flow (LDF) probes were inserted into parietal skull holes in anesthetized, splenectomized adult rats to measure cerebral RBC flow. HgbSS or HgbAA-RBCs (3 mls) were infused into rats after pretreatment with either the NO synthase (NOS) inhibitor, L-nitroarginine methyl-ester (L-NAME, 40 mg/kg IV), or its inactive isomer D-NAME. Mean arterial blood pressure, end-tidal CO2, and LDF were monitored for 30 minutes after the RBC infusion. The rats treated with L-NAME + HgbSS-RBCs(LN-SS group, n=9) experienced a significant decrease in LDF (p=0.002) as compared to controls at 20 min following the RBC infusion. In addition, 56% of the LN-SS group died prior to completion of the experiment. In the control groups, L-NAME + HgbAA-RBCs (n=6), D-NAME + HgbSS-RBCs (n=5), and D-NAME + HgbAA-RBCs (n=3), all rats completed the experimental protocol with no significant change in LDF and stable hemodynamics. In order to test thelocalized effect of NOS inhibition, three rats received a topical infusion of L-NAME through a cranial window that allowed direct visualization of RBC flow in the rat cerebral microvasculature. In two of these locally treated rats (67%), total cessation of cerebral blood flow was observed following infusion with HgbSS-RBCs while maintaining stable hemodynamics. We conclude that in the presence of HgbSS-RBCs, inhibition of NOS results in cerebral vaso-occlusion (i.e. stroke) in this rat model.