Nitric oxide (NO) is an essential signalling molecule for diverse physiological and disease processes. The current paradigm of how NO production is regulated focuses at the level of nitric oxide synthase (NOS), with respect to substrate and co-factor availability and the precise spatial and temporal arrangement of protein complexes. However, the respective unique or combined genetic deficiencies of the NOS isoforms exhibit relatively modest phenotypes in mice. Moreover, approaches targeted at modulating NOS activities have not successfully translated into different disease applications. All NOS isoforms are dependent on arginine as their sole substrate and interestingly, only one enzyme in mammals argininosuccinic lyase (ASL), can generate endogenous arginine. We propose that global regulation of NO production occurs earlier at the step of regulating arginine substrate availability within the cell. Until now, regulation at the level of arginine availability has been under-appreciated since arginine is readily available from external and dietary sources, irrespective of endogenous cellular production. However, extracellular arginine levels can affect NO production even though the intracellular levels of arginine are saturated.In humans, the natural history of argininosuccinic aciduria caused by deficiency of ASL shows systemic and chronic features that reflect in part global dysregulation of NO homeostasis. This led us to discover that ASL is required for the channelling of both endogenously synthesized arginine and exogenous arginine to NOS. By challenging the existing paradigm, I hypothesize that regulating ASL would allow us to characterize the cellular and molecular mechanisms underlying NO flux regulation at normal and pathological conditions, for therapeutic applications. This proposal is hence novel both in its concept but also in its approach that is based on targeting therapy for systemic disorders through regulating cellular metabolism.