Repetitive transcranial magnetic stimulation (rTMS) changes the function of the cortex. This study clarified the effects of acute rTMS treatment on extracellular serotonin (5-HT) concentrations in the rat prefrontal cortex (PFC) by using in vivo microdialysis methods. Each rat received acute rTMS treatment of the frontal brain at 500 stimuli from twenty trains applied at 25 Hz for 1 s at 1-min intervals between trains. Sham-treated rats received the same handling procedure and sound of the stimulator. Sham treatment increased the extracellular 5-HT levels compared with the non-treated group. However, rTMS treatment using the stimulation intensity of 110% motor threshold eliminated the increase in 5-HT levels induced by the sham treatment. Acute rTMS treatment of the frontal brain is related to the serotonergic neuronal system in the rat PFC, and it may have therapeutic implications for emotional disorders.