The SNARE proteins are essential components of the intracellular fusion machinery. It is thought that they form a tight four-helix complex between membranes, in effect initiating fusion. Most SNAREs contain a single coiled-coil region, referred to as the SNARE motif, directly adjacent to a single transmembrane domain. The neuronal SNARE SNAP-25 defines a subfamily of SNARE proteins with two SNARE helices connected by a longer linker, comprising also the proteins SNAP-23 and SNAP-29. We now report the initial characterization of a novel vertebrate homologue termed SNAP-47. Northern blot and immunoblot analysis revealed ubiquitous tissue distribution, with particularly high levels in nervous tissue. In neurons, SNAP-47 shows a widespread distribution on intracellular membranes and is also enriched in synaptic vesicle fractions. In vitro, SNAP-47 substituted for SNAP-25 in SNARE complex formation with the neuronal SNAREs syntaxin 1a and synaptobrevin 2, and it also substituted for SNAP-25 in proteoliposome fusion. However, neither complex assembly nor fusion was as efficient as with SNAP-25.