Regulated exocytosis is a crucial event for intercellular communication between neurons and astrocytes within the CNS. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex, composed of synaptobrevin 2, syntaxin and synaptosome-associated protein of 25 kDa or 23 kDa (SNAP25 or SNAP23), is essential in this process. It was reported that SNAP25 and SNAP23 have distinct roles in exocytotic release, where SNAP25, but not SNAP23, supports an exocytotic burst. It is not clear, however, whether this is due to the intrinsic properties of the ternary SNARE complex, containing either SNAP25 or SNAP23, or perhaps due to the differential association of these proteins with ancillary proteins to the complex. Here, using force spectroscopy, we show from single molecule investigations of the SNARE complex, that SNAP23A created a local interaction at the ionic layer by cuffing syntaxin 1A and synaptobrevin 2, similar to the action of SNAP25B; thus either of the ternary complexes would allow positioning of vesicles at a maximal distance of ∼13 nm from the plasma membrane. However, the stability of the ternary SNARE complex containing SNAP23A is less than half of that for the complex containing SNAP25B. Thus, differences in the stability of the two different ternary complexes could underlie some of the SNAP25/23 differential ability to control the exocytotic burst. © 2009 The Authors. Journal compilation © 2009 The Physiological Society.