Silencing-induced homeostatic plasticity is usually expressed as a change in the amplitude or the frequency of miniature postsynaptic currents. Here we report that, prolonged (approximately 24 h) silencing of mature (20-22 days in vitro) cultured hippocampal neurons using the voltage-gated sodium channel blocker tetrodotoxin (TTX) produced no effects on the amplitude or frequency of the miniature excitatory postsynaptic currents (mEPSCs). However, the silencing changed the intrinsic membrane properties of the neurons, resulting in an increased excitability and rate of action potentials firing upon TTX washout. Allowing neurons to recover in TTX-free recording solution for a short period of time after the silencing resulted in potentiation of mEPSC amplitudes. This form of activity-dependent potentiation is different from classical long-term potentiation, as similar potentiation was not seen in nonsilenced neurons treated with bicuculline to raise their spiking activity to the same level displayed by the silenced neurons during TTX washout. Also, the potentiation of mEPSC amplitudes after the recovery period was not affected by the N-methyl-d-aspartate receptor blocker d-2-amino-5-phosponopentanoic acid or by the calcium/calmodulin-dependent kinase II (CaMKII) inhibitor KN-62 but was abolished by the L-type calcium channel blocker nifedipine. We thus conclude that the potentiation of mEPSC amplitudes following brief recovery of spiking activity in chronically silenced neurons represents a novel form of metaplasticity that differs from the conventional models of homeostatic synaptic plasticity.