A body of evidence indicates that the production of adrenomedullin (ADM) in vivo is activated in states of inflammation. Our aim was to characterize the intracellular signaling pathways along which inflammation leads to a stimulation of ADM expression. For this purpose, we characterized the effects of inflammatory cytokines, tumor necrosis factor-&agr; (100 &mgr;g/L), interleukin-1&bgr; (20 &mgr;g/L), and interferon-&ggr; (0.5 U/L) on ADM gene expression in rat aortic vascular smooth muscle cells (AVSMCs). We found that inflammatory cytokines induced a time-dependent 12-fold upregulation of ADM mRNA in AVSMCs that was paralleled by a substantial increase in inducible NO synthase mRNA expression. The stimulatory effect of cytokines on ADM gene expression was attenuated by NO deprivation induced byN&ohgr;-nitro-l-arginine methyl ester (1 mmol/L) and was in part mimicked by the NO donorS-nitroso-N-acetylpenicillamine (100 &mgr;mol/L). The cGMP analog 8-bromo-cGMP (100 &mgr;mol/L) had no effect on ADM gene expression, and inhibition of cGMP production by 1H-oxodiazolo-quinoxalin-1 (ODQ, 200 &mgr;mol/L) was not able to abrogate the increase of ADM mRNA induced by NO donation usingS-nitroso-N-acetylpenicillamine (100 &mgr;mol/L). The significant induction of ADM gene expression by inflammatory cytokines and NO donation was also observed in mesangial cells, endothelial cells, and hepatocytes. These findings suggest that NO is a direct activator of ADM gene expression in a variety of cell types and that inflammatory cytokines stimulate ADM expression via both NO-dependent and -independent mechanisms. The stimulatory effect of NO appears to not be related to the classic guanylate cyclase–cGMP pathway.