Summary SNARE complex formation underlies intracellular membrane fusion in eukaryotic organisms; however, the factors regulating the SNARE assembly are not well understood. The neuronal SNARE complex is composed of synaptobrevin2, SNAP-25, and syntaxin1, the latter being under tight control by the cytosolic protein Munc18. We found that the inhibition of syntaxin1 by Munc18 both in nerve terminals and in defined in vitro reactions can be overcome by specific detergents. This serendipitous finding led us to screen biologically relevant fatty acids, revealing that unsaturated arachidonic and linolenic acids can stimulate Munc18-regulated SNARE complex formation in a direct manner. The direct effect of arachidonic acid on the syntaxin1/Munc18 complex suggests a mechanism for the activation of the SNARE assembly pathway and provides a lead for the further investigation of fatty acids that may regulate SNARE-mediated membrane fusion in eukaryotes.