Abstract: AIM　In order to explore the mechanism of action of melatonin to induce analgesia, the present study was undertaken to observe the effects of melatonin on the release of β-endorphin (β-Ep), norepinephrine (NE) and 5-hydroxytryptamine in rat brain. METHODS　With the measurement of pain threshold, push-pull perfusion technique and radioimmunoassay were used to determine the immunoreactive β-Ep content in the perfusate from the third ventricle of rat brain. The contents of 3-methoxy-4-hydroxyphenyl glycol (MHPG) and 5-hydroxyindole acetic acid in the microdialysate from rat brain were measured by techniques of in vivo microdialysis and high performance liquid chromatography with electrochemical detection. RESULTS The immunoreactive β-Ep content in the perfusate from the third ventricle of rat brain was increased significantly (P<0.05) following an intraperitoneal administration of 110 mg·kg^-1 of melatonin, with the increase of pain threshold. The MHPG and 5-HIAA contents in the microdialysate from the periaqueductal gray (PAG) or the hypothalamus were not changed after the administration of melatonin. CONCLUSION　Melatonin may promote the release of β-Ep in brain, which may be one of the mechanisms of the analgesic action of melatonin. The analgesic action of melatonin may not be related to the release of NE and 5-HT in the PAG or the hypothalamus.