The formation of chemical synapses in the mammalian brain involves complex pre- and postsynaptic differentiation processes. Presynaptically, the progressive accumulation of synaptic vesicles is a hallmark of synapse maturation in the neocortex [J Neurocytol 12 (1983b) 697]. In this study, we analyzed the functional consequences of presynaptic vesicle-pool maturation at central glutamatergic and GABAergic synapses. Using (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43) staining of recycling synaptic vesicles, we demonstrate a pronounced developmental increase in presynaptic vesicle accumulation during differentiation of neocortical neurons in culture. Using electrophysiological methods to study functional synaptic maturation, we found an improved recovery from hypertonic solution-induced depletion. As supported by the FM1-43 staining results, this change is most likely caused by a developmental increase in the number of reserve-pool vesicles. In addition, assuming a rapid reuse of freshly recycled vesicles, a developmental maturation of the endocytosis process may also contribute. The observed presynaptic maturation process occurred selectively at glutamatergic synapses, while GABAergic synapses did not show similar developmental alterations. Furthermore, we used high-frequency stimulation (HFS) of glutamatergic and GABAergic synapses to reveal the physiological consequences of reserve-pool maturation. As expected, recovery from HFS-induced depletion was incomplete at immature glutamatergic synapses and strongly improved during synapse maturation. Again, GABAergic synapses did not show similar developmental changes. Taken together, our study characterizes the functional consequences of a pronounced accumulation of reserve-pool vesicles occurring selectively at glutamatergic synapses.