Vesicular transporters accumulate neurotransmitters inside synaptic vesicles prior to exocytotic release. Vesicular glutamate transporters VGLUT1 and VGLUT2 are mainly expressed by subsets of neurons releasing the neurotransmitter glutamate, whereas VGLUT3 appears in populations of neurons usually associated with a different classical neurotransmitter. We have investigated physiological regulation of their subcellular localization. Using confocal microscopy, electron microscopy, and stereotactic injection of tracer dyes, we now bring new evidences for co-release of glutamate and GABA from the same nerve terminal. Immunoprecipitation experiments demonstrate that the specific vesicular transporters undergo differential targeting to specific subset of vesicles. Furthermore, investigation of these transporters in a pilocarpin-induced model for temporal lobe epilepsy reveals reorganization of the glutamatergic and GABAergic networks. Collectively, our studies show novel physiological and pathological roles of vesicular transporters.