The dynamic movement of synaptic vesicles is thought to be a key feature of synaptic transmission. However, the nature of synaptic vesicle mobility at nerve terminals still remains unclear. Here, we investigated synaptic vesicle mobility at the cultured calyx of Held, a giant brainstem presynaptic terminal, using real-time imaging of quantum dot-loaded single vesicles. Colloidal semiconductor nanocrystal quantum dots have been widely used as fluorescent probes due to their photochemical stability and high brightness. In our study, quantum dots were loaded into single vesicles and monitored with single particle tracking technique. Presynaptic terminal was activated by electrical stimulation and its electrical activity was measured by patch clamp recording from postsynaptic cell at the same time. This approach allows us to examine the movement of single vesicles in the presence and absence of presynaptic activity. Our results indicated that mobility of individual vesicles near the release site tended to increase in response to presynaptic activity. Thus, single vesicle dynamics can be investigated in real time imaging at nanometer scale resolution at mammalian central presynaptic terminals.