The release of neurotransmitter at a synapse occurs via the regulated fusion of synaptic vesicles with the plasma membrane. The fusion of the two lipid bilayers is mediated by a protein complex that includes the plasma membrane target soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (t-SNAREs), syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25), and the vesicle SNARE (v-SNARE), vesicle-associated membrane protein (VAMP). Whereas syntaxin 1A and VAMP are tethered to the membrane by a C-terminal transmembrane domain, SNAP-25 has been suggested to be anchored to the membrane via four palmitoylated cysteine residues. We demonstrate that the cysteine residues of SNAP-25 are not required for membrane localization when syntaxin 1A is present. Analysis of the 7 S and 20 S complexes formed by mutants that lack cysteine residues demonstrates that the cysteines are required for efficient SNARE complex dissociation. Furthermore, these mutants are unable to support exocytosis, as demonstrated by a PC12 cell secretion assay. We hypothesize that syntaxin 1A serves to direct newly synthesized SNAP-25 through the Golgi transport pathway to the axons and synapses, and that palmitoylation of cysteine residues is not required for targeting, but to optimize interactions required for SNARE complex dissociation.