Key points It is controversial whether glutamate can leak out of vesicles in the nerve terminal. To address this issue, we abolished vesicular glutamate uptake by washing out presynaptic cytosolic glutamate or by blocking vacuolar ATPase activity using bafilomycin A1. In the absence of vesicular glutamate uptake, both spontaneous and nerve‐evoked EPSCs underwent a rundown, suggesting that vesicular glutamate can leak out of vesicles. However, the rundown of evoked EPSCs was caused mainly by accumulation of unfilled vesicles after exocytic release of glutamate, suggesting a minor influence of glutamate leakage on synaptic transmission. Abstract Glutamate leaks out of synaptic vesicles when the transvesicular proton gradient is dissipated in isolated vesicle preparations. In the nerve terminal, however, it is controversial whether glutamate can leak out of vesicles. To address this issue, we abolished vesicular glutamate uptake by washing out presynaptic cytosolic glutamate in whole‐cell dialysis or by blocking vacuolar ATPase using bafilomycin A1 (Baf) at the calyx of Held in mouse brainstem slices. Presynaptic glutamate washout or Baf application reduced the mean amplitude and frequency of spontaneous miniature (m)EPSCs and the mean amplitude of EPSCs evoked every 10 min. The percentage reduction of mEPSC amplitude was much less than that of EPSC amplitude or mEPSC frequency, and tended to reach a plateau. The mean amplitude of mEPSCs after glutamate washout or Baf application remained high above the detection limit, deduced from the reduction of mEPSC amplitude by the AMPA receptor blocker 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione. Membrane capacitance measurements from presynaptic terminals indicated no effect of glutamate washout on exocytosis or endocytosis of synaptic vesicles. We conclude that glutamate can leak out of vesicles unless it is continuously taken up from presynaptic cytosol. However, the magnitude of glutamate leakage was small and had only a minor effect on synaptic responses. In contrast, prominent rundowns of EPSC amplitude and mEPSC frequency observed after glutamate washout or Baf application are likely to be caused by accumulation of unfilled vesicles in presynaptic terminals retrieved after spontaneous and evoked glutamate release.