This review focuses on recent advancements in the understanding of nitric oxide synthases (NOSs) over the past seven years. It covers the structural, functional, and inhibitory aspects of NOSs, highlighting significant progress in our knowledge of the enzyme's structure, from primary to quaternary levels. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) have provided insights into their binding sites for iron protoporphyrin IX (haem), biotin, L-arginine, and inhibitors. The mechanisms and products of the NOS reaction remain controversial, with emerging data suggesting one-electron redox cycling and multiple allosteric effects. Regulation of NOSs occurs at various levels, including gene transcription, covalent modification, and allosteric regulation. A wide range of NOS inhibitors have been identified, with diverse mechanisms of action, selectivity for different isoforms, and time-dependent effects. Selective inhibitors of iNOS and eNOS have shown potential in treating inflammatory conditions associated with iNOS activity. The review also discusses the role of biotin in NOS activity, the controversy over whether NOS directly synthesizes nitric oxide (NO) or nitroxyl (NO-), and the stoichiometry of the NOS reaction. The mechanisms of NO synthesis and the roles of the flavin and haem domains in NOS are explored, along with the diverse functions of the pterin cofactor. The review concludes by discussing feedback inhibition of NOS by NO and the regulation of NOSs in various tissues.This review focuses on recent advancements in the understanding of nitric oxide synthases (NOSs) over the past seven years. It covers the structural, functional, and inhibitory aspects of NOSs, highlighting significant progress in our knowledge of the enzyme's structure, from primary to quaternary levels. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) have provided insights into their binding sites for iron protoporphyrin IX (haem), biotin, L-arginine, and inhibitors. The mechanisms and products of the NOS reaction remain controversial, with emerging data suggesting one-electron redox cycling and multiple allosteric effects. Regulation of NOSs occurs at various levels, including gene transcription, covalent modification, and allosteric regulation. A wide range of NOS inhibitors have been identified, with diverse mechanisms of action, selectivity for different isoforms, and time-dependent effects. Selective inhibitors of iNOS and eNOS have shown potential in treating inflammatory conditions associated with iNOS activity. The review also discusses the role of biotin in NOS activity, the controversy over whether NOS directly synthesizes nitric oxide (NO) or nitroxyl (NO-), and the stoichiometry of the NOS reaction. The mechanisms of NO synthesis and the roles of the flavin and haem domains in NOS are explored, along with the diverse functions of the pterin cofactor. The review concludes by discussing feedback inhibition of NOS by NO and the regulation of NOSs in various tissues.