Neurotransmitter release proceeds by Ca²⁺-triggered, SNARE-complex-dependent synaptic vesicle fusion. After fusion, the ATPase NSF and its cofactors α- and βSNAP disassemble SNARE complexes, thereby recycling individual SNAREs for subsequent fusion reactions. We examined the effects of genetic perturbation of α- and βSNAP expression on synaptic vesicle exocytosis, employing a new Ca²⁺ uncaging protocol to study synaptic vesicle trafficking, priming, and fusion in small glutamatergic synapses of hippocampal neurons. By characterizing this protocol, we show that synchronous and asynchronous transmitter release involve different Ca²⁺ sensors and are not caused by distinct releasable vesicle pools, and that tonic transmitter release is due to ongoing priming and fusion of new synaptic vesicles during high synaptic activity. Our analysis of α- and βSNAP deletion mutant neurons shows that the two NSF cofactors support synaptic vesicle priming by determining the availability of free SNARE components, particularly during phases of high synaptic activity.