NO, produced by the endothelium, is a modulator of vascular inflammation. Traditionally, eNOS was believed to be the primary source of NO in the endothelium. However, recent data suggest an important role for nNOS in the endothelium, although little is known about factors regulating this novel eNOS. We examined the localization, regulation, and significance of endothelial nNOS in this study. Primary HUVECs were used as a model system. Inflammatory changes were induced by stimulation with TNF. We report that unlike eNOS, nNOS is predominantly localized to the nucleus of resting endothelial cells. This nNOS also contributed to basal NO production in the resting endothelium. Ablation of endothelial nNOS by pharmacological inhibition (using L-NPA) or siRNA further enhanced cytokine-mediated inflammatory responses, such as up-regulation of VCAM-1 and proinflammatory cytokines, as well as increased leukocyte recruitment. Based on these findings, we suggest a potential anti-inflammatory role of endothelial nNOS that can attenuate unopposed, proinflammatory cytokine actions. Our data indicate a novel location and an immunoregulatory role for nNOS in the endothelium.