The study investigates the interaction between endothelial nitric oxide synthase (eNOS) and caveolin-1, a key protein in caveolae. It demonstrates that eNOS directly interacts with caveolin-1 in both in vitro and in vivo settings. The binding occurs primarily in the scaffolding domain of caveolin-1 (amino acids 61-101) and to a lesser extent in the C-terminal tail (amino acids 135-178). This interaction leads to the inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. The study identifies the caveolin binding site within eNOS as being between amino acids 310 and 570. Mutagenesis of this site prevents caveolin-1 from suppressing NO release, indicating that caveolin-1 acts as a molecular chaperone that directly inactivates NOS. The findings suggest that the direct binding of eNOS to caveolin-1 and the targeting of eNOS to caveolae are distinct events that regulate NO production in endothelial cells. Additionally, the scaffolding domain of caveolin-3 can inhibit eNOS and nNOS, implying that caveolin-3 may negatively regulate NOS in cardiac myocytes and skeletal muscle. Caveolae, which are cholesterol- and sphingolipid-rich microdomains, are involved in various cellular functions, including signal transduction. The study highlights the role of caveolins as endogenous regulators of NOS and potential negative regulators of signal transduction. The study also shows that caveolin-1 can inhibit NO production from intact cells, and this inhibition is dependent on the caveolin binding domain (CBD) of eNOS. The results indicate that caveolin-1 negatively regulates eNOS in vivo, with the primary site of interaction being the predicted caveolin binding motif in eNOS. The study provides insights into the molecular mechanisms underlying the regulation of NOS by caveolins and highlights the importance of caveolae in signal transduction and NO production. The findings have implications for understanding the regulation of NO production in various cell types and tissues.The study investigates the interaction between endothelial nitric oxide synthase (eNOS) and caveolin-1, a key protein in caveolae. It demonstrates that eNOS directly interacts with caveolin-1 in both in vitro and in vivo settings. The binding occurs primarily in the scaffolding domain of caveolin-1 (amino acids 61-101) and to a lesser extent in the C-terminal tail (amino acids 135-178). This interaction leads to the inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. The study identifies the caveolin binding site within eNOS as being between amino acids 310 and 570. Mutagenesis of this site prevents caveolin-1 from suppressing NO release, indicating that caveolin-1 acts as a molecular chaperone that directly inactivates NOS. The findings suggest that the direct binding of eNOS to caveolin-1 and the targeting of eNOS to caveolae are distinct events that regulate NO production in endothelial cells. Additionally, the scaffolding domain of caveolin-3 can inhibit eNOS and nNOS, implying that caveolin-3 may negatively regulate NOS in cardiac myocytes and skeletal muscle. Caveolae, which are cholesterol- and sphingolipid-rich microdomains, are involved in various cellular functions, including signal transduction. The study highlights the role of caveolins as endogenous regulators of NOS and potential negative regulators of signal transduction. The study also shows that caveolin-1 can inhibit NO production from intact cells, and this inhibition is dependent on the caveolin binding domain (CBD) of eNOS. The results indicate that caveolin-1 negatively regulates eNOS in vivo, with the primary site of interaction being the predicted caveolin binding motif in eNOS. The study provides insights into the molecular mechanisms underlying the regulation of NOS by caveolins and highlights the importance of caveolae in signal transduction and NO production. The findings have implications for understanding the regulation of NO production in various cell types and tissues.