It is postulated that synchronous firing of cortical neurons plays an active role in cognitive functions of the brain. An important issue is whether pyramidal neurons in different cortical layers exhibit similar tendencies to synchronise. To address this issue, we performed intracellular and whole-cell recordings of regular-spiking pyramidal neurons in slice preparations of the rat motor cortex (18–45 days old) and analysed the phase response curves of these pyramidal neurons in layers 2/3 and 5. The phase response curve represents how an external stimulus affects the timing of spikes immediately after the stimulus in repetitively firing neurons. The phase response curve can be classified into two categories, type 1 (the spike is always advanced) and type 2 (the spike is advanced or delayed depending on the stimulus phase), and are important determinants of whether or not rhythmic synchronization of neuron pairs occurs. We found that pyramidal neurons in layer 2/3 tend to display type-2 phase response curves whereas those in layer 5 tend to exhibit type-1 phase response curves. The differences were prominent particularly in the gamma-frequency range (20–45 Hz). Our results imply that the layer-2/3 pyramidal neurons, when coupled mutually through fast excitatory synapses, may exhibit a much stronger tendency for rhythmic synchronization than layer-5 neurons in the gamma-frequency range.