The evolutionary conserved solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) fusion machinery is the operational unit in the release of neurotransmitters and hormones from excitable cells. The SNARE core complex consists of three proteins named SNAP-25 (synaptosomal-associated protein of 25 kD), syntaxin 1, and VAMP (vesicle-associated membrane protein)/synaptobrevin. Syntaxin 1 is, together with SNAP-25, localized to the plasma membrane, whereas VAMP/synaptobrevin is a component of secretory vesicles. In concert with the SNAREs, accessory factors govern the docking and priming of secretory vesicles prior to trans-SNARE complex formation and ultimately Ca2+-triggered fusion pore opening at the plasma membrane. The synaptic SNAP-25 protein exists as two closely related protein variants, named SNAP-25a and SNAP-25b. SNAP-25a and SNAP-25b are both encoded from a single copy gene and generated by obligate alternative splicing between two similar exon 5 sequences. Exon 5 spans a region of SNAP-25 that is subject to posttranslational palmitoylation and implicated in membrane anchoring of this cytosolic protein. The alternative splicing is strictly developmentally and neuroanatomically regulated, but the biological relevance of the distinct expression of these two similar protein variants is still a question of debate. However, recent findings in gene-targeted mouse mutants have started to unravel the importance that physiological levels of total SNAP-25 protein are present and, importantly, that this is accompanied by a balanced expression of SNAP-25a and SNAP-25b.