The formation of synaptic networks in the developing neocortex is thought to involve the activity-dependent stabilization of immature presynaptic transmitter release sites. However, the mechanisms involved in maturation of presynaptic function are not well understood at the subcellular and molecular level. We have now studied presynaptic developmental plasticity in layer 5B pyramidal neurons in acute slices of the somatosensory cortex of P7 and P14 mice. Evoked AMPA EPSCs were recorded using the whole-cell patch-clamp technique. Paired-pulse stimulation in slices of P7 mice revealed strong facilitation in about 25% of synapses tested. This indicates that a subset of synapses has a very low release probability (Pr). At P14 all glutamatergic synapses showed paired-pulse depression indicating a developmental maturation of the low Pr synapses. Upon pairing of presynaptic stimulation and postsynaptic depolarization at P7 paired-pulse facilitation was strongly reduced suggesting a NMDA receptor-dependent long-term increase in release probability. This result indicates that the developmental maturation of low Pr synapses is regulated by synaptic activity. In summary, we obtained evidence for presynaptically immature glutamatergic synapses in acute slices at P7 showing NMDA receptor-dependent plasticity.