Investigations of regulated S-nitrosylation and denitrosylation of vasorelevant proteins is a newly emergent area in vascular biology. We previously showed that monocrotaline pyrrole (MCTP)-induced megalocytosis of pulmonary arterial endothelial cells (PAECs), which underlies development of pulmonary arterial hypertension (PAH), was associated with a Golgi blockade characterized by trapping of diverse vesicle tethers, SNAREs and SNAPs in the Golgi; reduced trafficking of caveolin-1 (cav-1) and eNOS from the Golgi to the plasma membrane and decreased caveolar nitric oxide (NO). We have investigated whether N-ethylmaleimide sensitive factor (NSF), the ATPase involved in all SNARE-disassembly, might be the upstream target of MCTP and whether MCTP might regulate NSF by S-nitrosylation. Immunofluorescence microscopy and Golgi purification techniques revealed the discordant decrease of NSF by approximately 50% in Golgi membranes after MCTP despite increases in -SNAP, cav-1, eNOS and syntaxin-6. The NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) failed to affect initiation or progression of MCTP-megalocytosis despite reduction of 4,5-diaminofluorescein diacetate (DAF-2DA) fluorescence and inhibition of S-nitrosylation of eNOS as assayed using the biotin-switch method. Moreover, the latter assay not only revealed constitutive S-nitrosylation of NSF, eNOS, cav-1 and clathrin heavy chain (CHC) in PAECs, but also a dramatic 70-95% decrease in the S-nitrosylation of NSF, eNOS, cav-1 and CHC after MCTP. These data point to depletion of NSF from Golgi membranes as a mechanism for Golgi blockade after MCTP and to denitrosylation of vasorelevant proteins as critical to the development of endothelial-cell megalocytosis.