The consequence of reduced uptake of neurotransmitters into synaptic vesicles on synaptic transmission was examined in rat hippocampal slices and culture using bafilomycin A1 (Baf), a potent and specific blocker of the vacuolar-type (V-type) ATPase, which eliminates the driving force for the uptake of both glutamate and GABA into synaptic vesicles. After incubation with Baf, both the amplitude and frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were reduced in the slice preparation. Similar effects were seen with glutamatergic miniature excitatory postsynaptic currents (mEPSCs) and GABAergic mIPSCs from cultured neurons. This result indicates that vesicular content is reduced by Baf. The dramatic reduction in the frequency of mPSCs could result either from the exocytosis of empty vesicles or from a mechanism which prevents the exocytosis of depleted vesicles. Vesicle cycling was directly examined using confocal imaging with FM 1-43. In the presence of Baf, vesicles could still be endocytosed and they were released at the same probability as from control untreated synapses. Prolonged high-frequency electrical stimulation of synapses in culture failed to alter the amplitude of mEPSCs, suggesting that the filling of vesicles is rapid compared to the rate of vesicle recycling during repetitive synaptic stimulation. Profound release of glutamate with alpha-latrotoxin did cause a small, but reproducible, reduction in quantal size. These results indicate that decreasing the amount of glutamate and GABA in synaptic vesicles reduces quantal size. Furthermore, the probability of vesicle exocytosis appears to be entirely independent of the state of filling of the vesicle. However, even during high-frequency action potential-evoked release of glutamate, quantal size remained unchanged.