The reserve pool (RP) and readily releasable pool (RRP) of synaptic vesicles within presynaptic nerve terminals were physiologically differentiated into distinctly separate functional groups. This was accomplished in glutamate-ergic nerve terminals by blocking the glutamate transporter with TBOA (10µM) during electrical stimulation with either 40 Hz of 10 pulses within a train or 20 Hz or 50Hz continuous stimulation. The 50Hz continuous stimulation decreased the EPSP amplitude 60 minutes faster than for the 20Hz continuous stimulation in the presence of TBOA (p<0.05). There was no significant difference between the train stimulation and 20Hz continuous stimulation in the run down time in the presence of TBOA. After TBOA induced synaptic depression, the EPSPs were rapidly (<1 min) revitalized by exposure to serotonin (5-HT, 1µM) in every preparation tested (p<0.05). At this glutamate-ergic nerve terminal 5-HT promotes an increase probability of vesicular docking and fusion. Quantal recordings made directly at nerve terminals revealed smaller quantal sizes with TBOA exposure with a marked increase in quantal size as well as a continual appearance of smaller quanta upon 5-HT treatment after TBOA induced depression. Thus, 5-HT was able to recruit vesicles from the RP that were not rapidly depleted by acute TBOA treatment and electrical stimulation. The results support the notion that the RRP is selectively activated during rapid electrical stimulation sparing the RP; however, the RP can be recruited by the neuromodulator 5-HT. This suggests at least two separate kinetic and distinct regulatory paths for vesicle recycling within the presynaptic nerve terminal.