In this study, we observed quantal changes in single synapse excitatory postsynaptic currents to characterize N-methyl-D-aspartate receptor-mediated silent synapses between primary afferents and spinal substantia gelatinosa neurons. The failure rate of primary afferent quantal excitatory postsynaptic currents was lower at depolarized holding potentials than at hyperpolarized potentials. This lower failure rate at depolarized potentials was due to the activation of N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents. Pairing primary afferents with the postsynaptic depolarization induced a long-term decrease in the failure rate of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor-mediated excitatory postsynaptic currents, but did not alter the failure rate of N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents. Our findings suggest that pairing primary afferent with postsynaptic depolarization can convert silent substantia gelatinosa synapses to active synapses and the mechanism of this synaptic plasticity is associated with postsynaptic modification.