Stimulus-specific adaptation (SSA) is observed in many brain regions in humans and animals. SSA of cortical neurons has been proposed to accumulate through relays in ascending pathways. Here, we examined SSA at the synapse level using whole-cell patch-clamp recordings of primary cultured cortical neurons of the rat. First, we found that cultured neurons had high firing capability with 100-Hz current injection. However, neuron firing started to adapt to repeated electrically activated synaptic inputs at 10 Hz. Next, to activate different dendritic inputs, electrical stimulations were spatially separated. Cultured neurons showed similar SSA properties in the oddball stimulation paradigm compared to those reported in vivo. Single neurons responded preferentially to a deviant stimulus over repeated, standard stimuli considering both synapse-driven spikes and excitatory postsynaptic currents (EPSCs). Compared with two closely placed stimulating electrodes that activated highly overlapping dendritic fields, two separately placed electrodes that activated less overlapping dendritic fields elicited greater SSA. Finally, we used glutamate puffing to directly activate postsynaptic glutamate receptors. Neurons showed SSA to two separately placed puffs repeated at 10 Hz. Compared with EPSCs, GABAa receptor-mediated inhibitory postsynaptic currents showed weaker SSA. Heterogeneity of the synaptic inputs was critical for producing SSA, with glutamate receptor desensitization participating in the process. Our findings suggest that postsynaptic fatigue contributes largely to SSA at low frequencies.