L-arginine is the substrate for nitric oxide (NO) production by each of the 3 NO synthase (NOS) isoforms encoded by the mammalian genome. Despite the pivotal roles of NO in mammalian physiology and pathophysiology, the source of arginine for NO synthesis is not clearly defined. In this context, it is notable that cell types that do not have a complete urea cycle often possess the urea cycle enzymes argininosuccinate synthase and argininosuccinate lyase; together, these enzymes confer the ability to regenerate arginine from the NOS product, L-citrulline. Herein, the authors summarize evidence to support the view that argininosuccinate synthase and argininosuccinate lyase function in an arginine-citrulline cycle, providing a ready source of arginine for high-output NO synthesis. The arginine-citrulline cycle is induced in vascular cells by the same cytokines that trigger iNOS expression and provides the preferred source of substrate for NO production. Evidence suggests that argininosuccinate synthase activity is rate-limiting to high-output NO synthesis and, hence, represents a novel target for the treatment of pathophysiological conditions arising from NO overproduction.