To investigate the relationship between the prefrontal and hippocampal acetylcholine (ACh) systems and working memory, an in vivo microdialysis study was conducted. A group of rats was trained to perform a working memory task, delayed alternation, in an operant chamber for food reinforcement. The rats had to choose one of two response levers in an alternative manner in each trial, with a certain interval between trials. They had to remember which lever they chose in the previous trial without the assistance of external cues. Another group was trained to perform a reference memory task, cued alternation, in which the behavioral sequence was identical, but an external cue was provided. After stable behavior was established, a dialysis probe was implanted into the prefrontal cortex or the hippocampus of each rat. The extracellular concentration of ACh in the dialysates from the prefrontal cortex increased during performance of the delayed alternation task, while the hippocampal ACh showed a more distinct increase during performance of the cued alternation task. These results suggest that the prefrontal ACh is mainly related to working memory, whereas the hippocampal ACh is mainly related to reference memory.