The study investigates the interaction between endothelial nitric oxide synthase (eNOS) and caveolin-1, a resident coat protein of caveolae. Using in vitro and in vivo experiments, the researchers found that eNOS directly interacts with caveolin-1, particularly through its scaffolding domain (amino acids 61–101) and to a lesser extent through the C-terminal tail (amino acids 135–178). Peptides derived from these domains inhibited eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. The binding site for caveolin within eNOS was localized between amino acids 310 and 570, and mutation of this site blocked the negative regulation of NO release by caveolin-1. These findings suggest that caveolin-1 acts as a molecular chaperone, directly inactivating NOS, and that the interaction between eNOS and caveolin-1 may occur at different stages, regulating NO production in endothelial cells. Additionally, the study indicates that caveolin-3, a muscle-specific isoform, negatively regulates eNOS and nNOS in cardiac myocytes and skeletal muscle, respectively.The study investigates the interaction between endothelial nitric oxide synthase (eNOS) and caveolin-1, a resident coat protein of caveolae. Using in vitro and in vivo experiments, the researchers found that eNOS directly interacts with caveolin-1, particularly through its scaffolding domain (amino acids 61–101) and to a lesser extent through the C-terminal tail (amino acids 135–178). Peptides derived from these domains inhibited eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. The binding site for caveolin within eNOS was localized between amino acids 310 and 570, and mutation of this site blocked the negative regulation of NO release by caveolin-1. These findings suggest that caveolin-1 acts as a molecular chaperone, directly inactivating NOS, and that the interaction between eNOS and caveolin-1 may occur at different stages, regulating NO production in endothelial cells. Additionally, the study indicates that caveolin-3, a muscle-specific isoform, negatively regulates eNOS and nNOS in cardiac myocytes and skeletal muscle, respectively.