Activity plays important roles in the formation and maturation of synaptic connections. We examined these roles using solitary neocortical excitatory neurons, receiving only self-generated synaptic inputs, cultured in a microisland with and without spontaneous spike activity. The amplitude of excitatory postsynaptic currents (EPSCs), evoked by applying brief depolarizing voltage pulses to the cell soma, continued to increase from 7 to 14 days in culture. Short-term depression of EPSCs in response to paired-pulse or 10-train-pulse stimulation decreased with time in culture. These developmental changes were prevented when neurons were cultured in a solution containing tetrodotoxin (TTX). The number of functional synapses estimated by recycled synaptic vesicles with FM4-64 was significantly smaller in TTX-treated than control neurons. However, the miniature EPSC amplitude remained unchanged during development, irrespective of activity. Transmitter release probability, assessed by use-dependent blockade of N-methyl-D-aspartate receptor-mediated EPSCs with MK-801, was higher in TTX-treated than control neurons. Therefore, the activity-dependent increase in EPSC amplitude was mainly ascribed to the increase in synapse number, while activity-dependent alleviation of short-term depression was mostly ascribed to the decrease in release probability. The effect of activity blockade on short-term depression, but not EPSC amplitude, was reversed after 4 days of TTX removal, indicating that synapse number and release probability are controlled by activity in very different ways. These results demonstrate that activity regulates the conversion of immature synapses transmitting low-frequency input signals preferentially to mature synapses transmitting both low- and high-frequency signals effectively, which may be necessary for information processing in mature cortex.