AbstractGlutamate accumulation into synaptic vesicles is a vital step in glutamate synaptic transmission. In this study, we have explored the possibility that vesicular glutamate storage may be subject to some regulation. Synaptosomes were depolarized and subjected to [3H] glutamate under non-depolarizing conditions, and vesicular [3H] glutamate content was determined by a filter-based assay. We present evidence here that prolonged depolarization of synaptosomes leads to an increase in vesicular glutamate content. Induction of this enhanced state is time- and temperature-dependent. The enhanced state has two components, one readily reversible and the other long-lasting. The up-regulation of glutamate storage capacity could lead to an increase in quantal size and play a role in modulation of glutamate transmission efficiency.