We combined electron microscopy (EM), synaptic vesicle staining by a fluorescent marker FM1-43, photoconversion of the dye into an electron dense product, and electrical recordings of synaptic responses to investigate the distribution of reserve and recycling vesicles and its dependence upon stimulation in Drosophila motor boutons. We demonstrated that at rest vesicles are distributed over the periphery of the bouton, with the recycling and reserve pools being intermixed and the central core of the bouton being devoid of vesicles. Continuous high frequency stimulation following by a resting period mobilized the reserve vesicles into the recycling pool and, most notably, produced an increase in vesicle abundance. Recordings of synaptic activity from the temperature-sensitive endocytosis mutant shibire during continuous stimulation until complete depression, provided an independent estimate of the increase in vesicle abundance upon intense stimulation. EM analysis demonstrated that continuous stimulation produced an increase in the vesicle density, while during a subsequent resting period vesicles fill empty areas of the bouton, spreading towards its central core. Although the observed structural potentiation did not alter basal transmitter release, it produced an increased synaptic enhancement during high-frequency stimulation. The latter effect was not observed when the boutons were potentiated employing high frequency stimulation without a subsequent resting period. We concluded, therefore, that the newly formed vesicles replenish the reserve pool during a resting period following intense stimulation.