Endothelial dysfunction associated with elevated serum levels of tumor necrosis factor (TNF)- observed in diabetes, obesity and congenital heart disease results, in part, from the impaired production of endothelial nitric oxide (NO). Cellular NO production depends absolutely on the availability of arginine, the substrate for endothelial nitric oxide synthase (eNOS). In this report, evidence is provided demonstrating that treatment of bovine aortic endothelial cells with TNF- (10 ng/ ml) suppresses argininosuccinate synthase (AS), the rate-limiting step of the citrulline-NO cycle. Western blot and real time RT-PCR demonstrated a significant and dose-dependent reduction of AS protein and mRNA when treated with TNF- with a corresponding decrease in NO production. Reporter gene analysis demonstrated that TNF- suppresses the AS proximal promoter, and EMSA analysis showed reduced binding to three essential Sp1 elements. Inhibitor studies showed that the repression of AS expression by TNF- may be mediated, in part, via the NFB signaling pathway. These findings also demonstrated that TNF- coordinately down-regulates AS and eNOS expression, in part through a similar mechanism, resulting in severely impaired endothelial NO production. The down-regulation of AS by TNF- is an added insult to endothelial function due to the important role of AS in NO production and in endothelial viability.