Although cellular levels of arginine greatly exceed the apparent K m for endothelial nitric-oxide synthase, current evidence suggests that the bulk of this arginine may not be available for nitric oxide (NO) production. We propose that arginine regeneration, that is the recycling of citrulline back to arginine, defines the essential source of arginine for NO production. To support this proposal, RNA interference analysis was used to selectively reduce the expression of argininosuccinate synthase (AS), because the only known metabolic role for AS in endothelial cells is in the regeneration of L-arginine from L-citrulline. Western blot analysis demonstrated a significant and dose-dependent reduction of AS protein as a result of AS small interfering RNA treatment with a corresponding diminished capacity to produce basal or stimulated levels of NO, despite saturating levels of arginine in the medium. Unanticipated, however, was the finding that the viability of AS small interfering RNA-treated endothelial cells was significantly decreased when compared with control cells. Trypan blue exclusion analysis suggested that the loss of viability was not because of necrosis. Two indicators, reduced expression of Bcl-2 and an increase in caspase activity, which correlated directly with reduced expression of AS, suggested that the loss of viability was because of apoptosis. The exposure of cells to an NO donor prevented apoptosis associated with reduced AS expression. Overall, these results demonstrate the essential role of AS for endothelial NO production and cell viability.Nitric oxide (NO) 1 is an important modulator for a wide range of functions including vasodilation of blood vessels, immune system function, angiogenesis, inhibition of leukocyte adhesion and platelet aggregation, gene regulation, and apoptosis (1-3). Moreover, NO has a dual role in cell viability depending on the tissue type and concentration. Either very high or very low concentrations of NO may induce cell death, whereas basal concentrations may inhibit apoptosis (4 -7). Previous work has shown that NO protects against serum starvation-(8), H 2 O 2 -(9), TNF-␣-(10), and oxidized low density lipoprotein-induced apoptosis (11, 12) in endothelial cells.Argininosuccinate synthase (AS), the rate-limiting step (13) in the regeneration of arginine from citrulline, catalyzes the synthesis of argininosuccinate, AMP, and inorganic pyrophosphate from citrulline, ATP, and aspartate. Argininosuccinate is then cleaved by argininosuccinate lyase (AL) to produce Larginine and fumarate. In the liver, AS and AL function together as components of the urea cycle, ultimately to form arginine from citrulline. Although the expression of AS and AL in the liver is high, both enzymes are found in most mammalian tissues, although at significantly lower levels. The discovery of arginine-derived NO, catalyzed by nitric-oxide synthases (NOSs), revealed a second role for AS and AL (1-3). Together with NOS, they function as part of a citrulline-NO cycle where AS and AL convert citrul...