© 2019, eLife Sciences Publications Ltd. All right reserved. The number of neurotransmitter-filled vesicles released into the synaptic cleft with each action potential dictates the reliability of synaptic transmission. Variability of this fundamental property provides diversity of synaptic function across brain regions, but the source of this variability is unclear. The prevailing view is that release of a single (univesicular release, UVR) or multiple vesicles (multivesicular release, MVR) reflects variability in vesicle release probability, a notion that is well-supported by the calciumdependence of release mode. However, using mouse brain slices, we now demonstrate that the number of vesicles released is regulated by the size of the readily-releasable pool, upstream of vesicle release probability. Our results point to a model wherein protein kinase A and its vesicle-associated target, synapsin, dynamically controls release site occupancy to dictate the number of vesicles released without altering release probability. Together these findings define molecular mechanisms that control MVR and functional diversity of synaptic signaling.