N-Terminal myristoylation and thiopalmitoylation of the endothelial isoform of nitric oxide synthase (eNOS) are required for targeting the enzyme to specialized signal-transducing microdomains of plasma membrane termed caveolae. We have previously documented that the subcellular localization of eNOS is dynamically regulated by agonists such as bradykinin, which promotes enzyme depalmitoylation and translocation from caveolae. More recently, we have shown that association of eNOS with caveolin, the principal structural protein in caveolae, leads to enzyme inhibition, in a reversible process modulated by Ca²⁺−calmodulin (CaM). We now report studies of the respective roles of acylation and caveolin interaction for regulating eNOS activity. Using eNOS truncation and deletion mutants expressed in COS-7 cells, we have identified an obligatory role for the N-terminal half of eNOS in stabilizing its association with caveolin. By exploring the differential effects of detergents (CHAPS vs octyl glucoside), we have shown that this direct interaction between both proteins is facilitated by, but does not require, eNOS acylation, and, importantly, that treatment of intact aortic endothelial cells with the calcium ionophore A23187 leads to the rapid disruption of the eNOS−caveolin complexes. Finally, using transiently transfected COS-7 cells, we have observed that the myristoylation-deficient cytosol-restricted eNOS mutant (myr^-) as well as the cytosolic fraction of the palmitoylation-deficient eNOS mutant (palm^-) may both interact with caveolin; this association also leads to a marked inhibition of enzyme activity, which is completely reversed by addition of calmodulin. We conclude that the regulatory eNOS−caveolin association is independent of the state of eNOS acylation, indicating that agonist-evoked Ca²⁺/CaM-dependent disruption of the caveolin−eNOS complex, rather than agonist-promoted depalmitoylation of eNOS, relieves caveolin's tonic inhibition of enzyme activity. We therefore propose that caveolin may serve as an eNOS chaperone regulating NO production independently of the enzyme's residence within caveolae or its state of acylation.