The expression of inducible nitric oxide synthase (NOS2) in glial cells is inhibited by neurotransmitters such as norepinephrine (NE) which elevate cAMP levels. We examined the molecular basis for this effect using a 2.2-kb fragment of the rat NOS2 promoter transfected into rat C6 glioma cells. Promoter activation (up to six-fold) by lipopolysaccharide (LPS) and interferon-γ (IFNγ) was reduced by NE, which alone had no effect. However, a promoter construct extending to bp −130 and containing the proximal nuclear factor-kappa B (NF-κB) binding site was minimally activated by LPS and cytokines, but activated up to three-fold by NE. Deletion analysis identified a 27-bp region (bp −187 to −160) as critical for mediating this suppressive effect. This region also enhanced promoter activation by LPS and cytokines, and prevented activation by NE alone. Gel shift analysis revealed constitutive binding to this region, and induction by NE of additional complexes which could be blocked by an antibody against CREB. NE also increased levels of the IκBα protein which could contribute to its suppressive effects. These results identify a critical role for this 27-bp region in regulation of NOS2 promoter activation and suppression by cAMP.