The presence of myristoylated endothelial nitric oxide synthase (eNOS) in cytosolic fractions of bovine aortic endothelial cells (BAEC) suggests that N-myristoylation of eNOS is not sufficient for membrane localization. Therefore, we examined if posttranslational palmitoylation was another molecular signal for the membrane attachment of eNOS. Metabolic labeling showed incorporation of [3H]palmitic acid into the membrane-bound, but not the cytosolic, form of eNOS. Fatty acid analysis demonstrated the labeled fatty acid incorporated into eNOS was palmitate, linked via a hydroxylamine-labile thioester bond. Biosynthesis and turnover studies show that the turnover of palmitate is much faster than the protein itself. However, the rates of palmitoylation and depalmitoylation were not affected by bradykinin or ionomycin treatment of BAEC. To examine the contribution of palmitoylation to the membrane association of eNOS, we mutated cysteine-15 and -26. Both mutations markedly diminished palmitoylation of eNOS, but did not significantly alter its membrane association. Additionally, [3H]palmitic acid was not incorporated into nonmyristoylated mutant eNOS (G2A eNOS), suggesting that myristoylation is necessary for subsequent palmitoylation of the enzyme. Taken together, our data imply that palmitoylation does not play a major role in membrane association of eNOS and other amino acid sequences, in conjunction with N-myristoylation, are necessary and sufficient for membrane association of the enzyme.