Using a novel approach to measure exocytosis in vitro from semi-intact synaptosomes, we establish that the Ca²⁺-dependent release of glutamate requires cytosolic factors for mobilization from the reserve pool. The cytosolic activity for glutamate release was not satisfied by CAPS, a soluble component required for norepinephrine (NE) release. Moreover, the CAPS-independent glutamate release from synaptic vesicles (SVs) was 200-fold less sensitive to Ca²⁺ than that required for dense core vesicles (DCVs). The differential regulation of exocytosis by CAPS, Ca²⁺, and potential novel cytosolic factor(s) suggests that the docking and fusion machinery controlling DCVs has diverged from that regulating glutamate-containing SVs.