Synaptic activity in the medial prefrontal cortex (mPFC) is fundamental for higher cognitive functions such as working memory. The present study shows that small conductance (SK) calcium-activated potassium channels attenuate excitatory synaptic transmission at layer 2/3 and layer 5 inputs to layer 5 pyramidal neurons in the mPFC. SK channels are located postsynaptically at synapses where they are activated during synaptic transmission by calcium influx through NMDA receptors, L-type calcium channels, R-type calcium channels and by calcium release from IP(3)-sensitive stores. Removal of the SK channel-mediated shunt of synaptic transmission reveals significant NMDA receptor-mediated activation during basal synaptic transmission, which is greater at layer 5 inputs (approximately 30%) than at layer 2/3 inputs (approximately 20%). These findings show that interactions between NMDA receptors, SK channels and voltage-gated calcium channels play a critical role in regulating excitatory synaptic transmission in layer 5 pyramidal neurons in the mPFC.