At the excitatory synapse neurotransmitter glutamate is released from synaptic vesicles. After exocytosis vesicles are retrieved by endocytosis and recycled for reuse, thereby maintaining synaptic transmission. During recycling vesicles are refilled with glutamate by vesicular glutamate transporters (VGLUTs), using electrochemical gradient produced by vacuolar-type H+-ATPase (V-ATPase). Multiple recycling mechanisms of different speeds are thought to operate in the nerve terminal. However, the speed of vesicle refilling with glutamate is not known. Here we manipulated vesicular glutamate directly in the nerve terminal, depleting it by whole-cell dialysis and refilling it by the photolysis of caged glutamate. Vesicle refilling, monitored by postsynaptic currents, was abolished by blocking VGLUT or V-ATPase, and attenuated by increasing cytosolic Cl− concentrations. The refilling time constant ranged 2-13 s depending upon the magnitude of refilling, with 100% refilling time constant being 18 s. This rate is faster than the "full-fusion"-type slow recycling time (1 min), but slower than "kiss-and-run"-type fast recycling time (<1s). We conclude that transmitter glutamate can fully refill vesicles recycled via slow, but not fast, pathways. [J Physiol Sci. 2006;56 Suppl:S87]