We have studied glutamate release from synaptic vesicles in permeabilized synaptosomes, which were preloaded with [3H]glutamate in an ATP-dependent manner. The release was found to be calcium-dependent and to require a heat-labile cytosolic macromolecule factor for maximum activity. Maximal release occurred at 5 microM free Ca2+ and within 5 min. Of the other divalent cations tested, only barium stimulated release of vesicular glutamate. The release was inhibited by N-ethylmaleimide. These results are characteristic of exocytotic release of monoamines and peptides observed in endocrine systems, and constitute direct evidence for the notion that calcium-dependent release of glutamate originates from the vesicular pool.