We have reported that long-term potentiation (LTP) can be reliably induced in motor cortex of adult, freely moving rats by the application of spaced and repeated high frequency stimulating trains to the white matter. In the present study, we monitored field potentials (FPs) and LTP in both layer II/III and V in coronal slices of motor cortex taken from implanted control and previously potentiated Long–Evans rats. The baseline FP amplitudes were decreased in layer II/III, and the amplitude of small spikes was significantly increased in layer V in slices from previously potentiated rats compared to unpotentiated control rats. In response to high frequency stimulation applied to the slice itself, both implanted control and previously potentiated rats showed similar levels of LTP in layer II/III. LTP could not be induced in layer V. These results show that layer II/III and V respond differently to high frequency stimulation in vitro. In addition, layer II/III responds very differently in slice compared to chronic preparations.