We investigated quantal release and ultrastructure in the neuromuscular junctions of synapsin II knockout (Syn II KO) mice. Synaptic responses were recorded focally from the diaphragm synapses during electrical stimulation of the phrenic nerve. We found that synapsin II affects transmitter release in a Ca2+ dependent manner. At reduced extracellular Ca2+ (0.5 mM), Syn II KO mice demonstrated a significant increase in evoked and spontaneous quantal release, while at the physiological Ca2+ concentration (2 mM), quantal release in Syn II KO synapses was unaffected. Protein kinase inhibitor H7 (100ìm) suppressed quantal release significantly stronger in Syn II KO synapses than in WT, indicating that Syn II KO synapses may compensate for the lack of synapsin II via a phosphorylation-dependent pathway. Electron microscopy analysis demonstrated that the lack of synapsin II results in an approximately 40% decrease in the density of synaptic vesicles in the reserve pool, while the number of vesicles docked to the presynaptic membrane remained unchanged. Synaptic depression in Syn II KO synapses was slightly increased, which is consistent with the depleted vesicle store in these synapses. At reduced Ca2+ frequency facilitation of synchronous release was significantly increased in Syn II KO, while facilitation of asynchronous release was unaffected. Thus, at the reduced Ca2+ concentration, synapsin II suppressed transmitter release and facilitation. These results demonstrate that synapsin II can regulate vesicle clustering, transmitter release, and facilitation.