At the synapse, SNAP-25, along with syntaxin/HPC-1 and synaptobrevin/VAMP, forms SNARE (soluble NSF attachment protein receptor) complexes that are thought to catalyze membrane fusion. Results from neuronal cultures of synaptobrevin-2 knock-out (KO) mice showed that loss of synaptobrevin has a more severe effect on calcium-evoked release than on spontaneous release or on release evoked by hypertonicity. In this study, we recorded neurotransmitter release from neuronal cultures of SNAP-25 KO mice in order to determine whether they share this property. In neurons lacking SNAP-25, as those deficient in synaptobrevin-2, we found that approximately 10-12% of calcium-independent excitatory and inhibitory neurotransmitter release persisted. However, in contrast to synaptobrevin-2 knockouts, this remaining readily releasable pool in SNAP-25 deficient synapses was virtually insensitive to calcium-dependent evoked stimulation. While field stimulation reliably evoked neurotransmitter release in synaptobrevin-2 KO neurons, responses were rare in neurons lacking SNAP-25, and unlike synaptobrevin-2 deficient synapses, SNAP-25 deficient synapses did not exhibit facilitation of release during high-frequency stimulation. This severe loss of evoked exocytosis was matched by a reduction, but not a complete loss, of endocytosis during evoked stimulation. Moreover, synaptic vesicle turnover probed by FM-dye uptake and release during hypertonic stimulation was relatively unaffected by the absence of SNAP-25. This last difference indicates that in contrast to synaptobrevin, SNAP-25 does not directly function in endocytosis. Together, these results suggest that SNAP-25 has a more significant role in calcium-secretion coupling than synaptobrevin-2.