Endothelial nitric-oxide synthase (eNOS) undergoes a complex pattern of post-translational modifications that regulate its activity. We have recently reported that eNOS is constitutively S-nitrosylated in endothelial cells and that agonists promote eNOS denitrosylation concomitant with enzyme activation (Erwin, P. A., Lin, A. J., Golan, D. E., and Michel, T. (2005), J. Biol. Chem. 280, 19888-19894). In the present studies, we use mass spectrometry to confirm that the zinc-tetrathiolate cysteines of eNOS are S-nitrosylated. eNOS targeting to the plasma membrane is necessary for enzyme S-nitrosylation, and we report that translocation between cellular compartments is necessary for dynamic eNOS S-nitrosylation. We transfected cells with cDNA encoding wild-type eNOS, which is membrane-targeted, or with acylation-deficient mutant eNOS (Myr-), which is expressed solely in the cytosol. While wild-type eNOS is robustly S-nitrosylated, we found that S-nitrosylation of the Myr- eNOS mutant is nearly abolished. When we transfected cells with a fusion protein in which Myr- eNOS is ligated to the CD8-transmembrane domain (CD8-Myr-), we found that CD8-Myr- eNOS, which does not undergo dynamic subcellular translocation, is hypernitrosylated relative to wild-type eNOS. Furthermore, we found that when endothelial cells transfected with wild-type or CD8-Myr- eNOS are stimulated with eNOS agonist, only wild-type eNOS is denitrosylated; CD8-Myr- eNOS S-nitrosylation is unchanged. These findings indicate that subcellular targeting is a critical determinant of eNOS S-nitrosylation. Finally, we show that eNOS S-nitrosylation can be detected in intact arterial preparations from mouse and that eNOS S-nitrosylation is a dynamic agonist-modulated process in intact blood vessels. These studies suggest that receptor-regulated eNOS S-nitrosylation may represent an important determinant of NO-dependent signaling in the vascular wall.