The nitric oxide synthases (NOS) comprise a family of enzymes which differ in primary structure, biological roles, subcellular distribution, and post-translational modifications. The endothelial nitric oxide synthase (ec-NOS) is unique among the NOS isoforms in being modified by N-terminal myristoylation, which is necessary for its targeting to the endothelial cell membrane. The subcellular localization of the ecNOS, but not enzyme myristoylation, is dynamically regulated by agonists such as bradykinin, which promote ecNOS translocation from membrane to cytosol, as well as enhancing enzyme phosphorylation. Using transiently transfected endothelial cells, we now show that a myristoylation-deficient mutant ecNOS undergoes phosphorylation despite restriction to the cytosol, suggesting that phosphorylation may be a consequence rather than a cause of ecNOS translocation. We therefore explored whether other post-translational modifications might regulate ecNOS targeting and now report that ecNOS is reversibly palmitoylated. Biosynthetic labeling of endothelial cells with [3H]palmitic acid followed by immunoprecipitation of ecNOS revealed that the enzyme is palmitoylated; the label is released by hydroxylamine, consistent with formation of a fatty acyl thioester, and authentic palmitate can be recovered from labeled ecNOS following acid hydrolysis. Importantly, pulse-chase experiments in endothelial cells biosynthetically labeled with [3H]palmitate show that bradykinin treatment promotes ecNOS depalmitoylation. We conclude that ecNOS palmitoylation is dynamically regulated by bradykinin and propose that depalmitoylation of the enzyme may result in its cytosolic translocation and subsequent phosphorylation.