Nitric oxide is a ubiquitous molecule implicated in a variety of biological processes. The specific action of nitric oxide depends on its enzymatic sources, namely neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), each having distinct tissue localization. Conventional pharmacological antagonists could not distinguish these enzymes or provide models of chronic nitric oxide depletion in whole animals. Several lines of knockout mice have been generated to distinguish the roles of nitric oxide from each enzyme: nitric oxide from nNOS is a major inhibitory neurotransmitter, nitric oxide from eNOS regulates blood flow under physiological conditions, and nitric oxide from iNOS causes hypotension during severe inflammatory conditions. Moreover, the nitric oxides from each isoform have different roles in tissue injury and inflammation. Studies of NOS-deficient animals have also identified redundant and compensatory pathways and revealed the consequences of life-long deficiency of these enzymes. The nNOS-deficient mice develop gastric dilation and stasis, the eNOS-deficient mice develop hypotension and lack vasodilatory responses to injury, and iNOS-deficient mice are more susceptible to inflammatory damage but more resistant to septic shock.