The ubiquitous process of membrane fusion in eukaryotes is executed by a host of essential conserved factors, including membrane associated SNARE proteins such as syntaxins. SNARE complex formation brings membranes into close apposition for fusion. The lipid metabolism of the surrounding bilayer, and highly conserved cytosolic components such as Munc18, are also important. The interaction of Munc18 with syntaxin is essential for exocytosis. Knock-out of Munc18 results in dramatically decreased secretion (Verhage et al, 2000) yet paradoxically, initial in vitro experiments suggested that the syntaxin/Munc18 interaction is incompatible with SNARE assembly. It was recently found that Munc18 inhibition of neuronal syntaxin1 can be overcome by arachidonic acid (Rickman and Davletov, 2005), suggesting that this common second messenger disrupts syntaxin/Munc18 association. We now show that arachidonic acid can stimulate syntaxin1 in the absence of Munc18 (Figure 1). Although arachidonic acid relieves inhibition of Munc18-bound syntaxin (Fig2A), it cannot dissociate this syntaxin1/Munc18 complex (Fig2B). Upon arachidonic acid-induced syntaxin1/SNAP25 association, Munc18 remains attached to syntaxin1, and a Munc18/syntaxin/SNAP25 complex is formed (Fig2C). We show for the first time that neuronal SNAREs can be isolated from brain membranes assembled with endogenous Munc18 (Fig2D,E) consistent with Munc18’s role in downstream vesicle docking and fusion events (Toonen et al, 2006).