At vertebrate central synapses, it has been demonstrated that a resting pool of synaptic vesicles (SVs) exists that normally does not participate in SV release and recycling. It remains unclear whether SVs within the resting pool are capable of mobilization and fusion. Here, we combine live imaging of SV exo- and endocytosis using pH-sensitive GFP (synapto-pHluorins) with pharmacological and genetic manipulations of the SV cycle at the Drosophila NMJ. We demonstrate that a resting pool of SVs exists at this synapse that encompasses 30-41% of the total SV pool. Under conditions of endocytic blockade, using a temperature-sensitive dynamin mutation, the resting pool of SVs can be mobilized and released. We present a model for the presence of a resting pool of SVs that does not require molecular specification of a subpopulation of SVs.