We previously demonstrated that norepinephrine (NE) inhibits induction of the calcium-independent isoform of nitric oxide synthase (NOS-2) in primary rat astrocyte cultures. However, the molecular mechanisms underlying this effect are unknown. In C6 cells and astrocytes, NE suppressed both cytokine- and lipopolysaccharide (LPS)-dependent nitrite accumulation, an index of NOS-2 activity. NE reduced the steady-state levels of NOS-2 mRNA induced by LPS plus cytokines but did not decrease NOS-2 mRNA stability or inhibit activation or subunit composition of transcription factor nuclear factor kappaB, which is necessary for NOS-2 induction. In C6 cells stably transfected with a 1,588-bp mouse NOS-2 promoter, NE reduced LPS plus cytokine-induced reporter gene expression, suggesting inhibition of NOS-2 promoter activity. In contrast, suppression was lost when a truncated 85-bp NOS-2 promoter was used, and in these cells NE potentiated reporter gene expression, alone or in the presence of LPS and cytokines. These results suggest that the suppressive effects of NE are due to modification of transcription factor activity in a region located between -1,588 and -85 of the NOS-2 promoter and may help explain observations that in some cells cyclic AMP can potentiate, rather than suppress, NOS-2 expression.