The aim of the present study was to investigate the role of nucleus raphe obscurus (NRO) in regulating the motility of sphincter of Oddi (SO). After fasting about l8~24 h, the rabbits were anesthetized with urethane (1.0 g/kg), and the myoelectric signals of SO were induced by a pair of copper electrodes inserted into the subsera. The results of microinjection of various drugs into NRO are as follows. After glutamate (340 mmol/L, 0.1 microl) was injected, activity of SO was excited. With microinjection of GABA (1 mol/L,0.1 microl), the spike burst of SO was inhibited. Following microinjection of ketamine (180 mmol/L, 0.1 microl), a kind of N-methyl-D-aspartate (NMDA) receptor antagonist, SO motility was inhibited and the effect of glutamate was abolished. Injection of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (2 mmol/L, 0.1 microl), a non-NMDA receptor antagonist, also excited the myoelectric activity of SO, but did not inhibit the effect of glutamate. The effect of glutamate was abolished by intravenous injection of atropine (0.2 mg/kg) or bilateral vagotomy, but not by injection of phentolamine (1.5 mg/kg) or propronalol (1.5 mg/kg), or by transection of the spinal cord. The above results indicate that NRO mediation of SO activity is due to the effect of glutamate on the NMDA receptors in the nucleus, the output of which is sent through vagal nerve and peripheral M cholinergic receptor to exert excitation of gallbladder motility.