Exocytosis is generally triggered by a rise in the intracellular free calcium concentration. This calcium-induced secretion is also subject to modulation by a variety of signalling mechanisms. The most widely studied and best understood of these is protein phosphorylation, which has been recognised for many years to be a near universal modulator of regulated exocytosis. More recently, nitric oxide-induced protein S-nitrosylation has emerged as an important post-translational modification affecting exocytosis in a variety of cell types, including neurons. Nevertheless, there is relatively little mechanistic understanding of how post-translational modification of the protein machinery for exocytosis can lead to the observed cellular effects. Formation of a ternary SNARE complex between syntaxin, SNAP-25 and VAMP is essential for neuroexocytosis and is believed to directly drive the membrane fusion process. Complex formation occurs sequentially, proceeding via an initial syntaxin-SNAP-25 heterodimer intermediate, to which VAMP subsequently interacts. The ability of syntaxin to form such a binary complex is in turn dependent on its SNARE motif being accessible, thus requiring the adoption of a so-called ‘open’ conformation. In contrast, in the ‘closed’ conformation, the SNARE motif of syntaxin is sequestered in an intramolecular helical bundle that prevents binding to SNAP-25, but allows binding to Munc18. As syntaxin is thought to exist in equilibrium between open and closed conformations, and as the formation of the initial syntaxin/SNAP-25 heterodimer is the rate-limiting step in assembly of the ternary SNARE complex, this represents a potential point of regulation by signalling mechanisms. Here we report that post-translational modification of syntaxin can regulate this process. In protein interaction assays in vitro, nitrosylation of a single cysteine residue in syntaxin inhibits binding to Munc18-1, but facilitates binding to SNAP-25 and VAMP to form the ternary SNARE complex. This effect is apparently mediated by a conformational change in syntaxin, as it induces alterations in protease sensitivity that mimic those seen in the previously characterised open mutant syntaxin. Consistent with this notion, expression of a cysteine mutant syntaxin construct designed to mimic the nitrosylated state alters the release kinetics of single vesicle exocytosis events in a similar manner to the open mutant. Taken together, our data suggest that nitrosylation of syntaxin may be a regulatory switch that helps the protein to adopt a more ‘open’ conformation, thereby facilitating SNARE complex formation and hence membrane fusion.