To determine whether the rat medial prefrontal cortex (PFC) is involved in acquiring operant learning, we observed changes in extracellular concentration of dopamine (DA) and acetylcholine (ACh) in the rat medial PFC during lever-press acquisition (acquisition group) or retrieval (retention group) using in vivo microdialysis. We found that DA or ACh elevation related to acquisition occurred. DA elevation was observed in the acquisition group only. These results indicate that the medical PFC is related to acquisition, and suggest that interaction between DA and ACh may be involved in learning acquisition.