In this study, we evaluated whether different frequencies of electroacupuncture (EA) modified the activities of serotonergic neurons in the dorsal raphe (DR) and raphe magnus (RMg) using double labeling immunohistochemistry for Fos and serotonin. The results demonstrated that both high and low frequency EA increased the colocalization between Fos and serotonin in the DR, not in RMg as compared with anesthesia control. In addition, high frequency EA more potently increased the serotonergic activity in the DR rather than low frequency EA, suggesting that serotonergic pathway from the DR plays an important role in the high frequency EA analgesia.