Whether glutamate is released rapidly, in an all-or-none manner, or more slowly, in a regulated manner, is a matter of debate. We analyzed the time course of excitatory postsynaptic currents (EPSCs) at glutamatergic neuromuscular junctions of Drosophila and found that the decay phase of EPSCs was protracted to a variable extent. The protraction was more pronounced in evoked and spontaneous quantal EPSCs than in action potential-evoked multiquantal EPSCs; reduced in quantal EPSCs from endophilin null mutants, which maintain release via kiss-and-run; and dependent on synaptotagmin isoform, calcium, and protein phosphorylation. Our data indicate that glutamate is released from individual synaptic vesicles for milliseconds through a fusion pore. Quantal glutamate discharge time course depends on presynaptic calcium inflow and the molecular composition of the release machinery.