The microdialysis technique was applied to detect the changes in the activity of acetylcholine (ACh) neurons in the rat brain. The effects of intraperitoneal (i.p.) injection of toluene on the amount of ACh release from the nerve terminals of the brain cholinergic neurons were investigated in freely moving rats. In the striatum, injection of toluene decreased the extracellular concentration of ACh in a dose dependent manner in the range 200 to 2,000 mg/kg. Similar effects of toluene on ACh release were observed in the hippocampus after i.p. administration. The increases in ACh content in brain homogenate after i.p. injection of toluene seemed to be caused by the decreased release of ACh from cholinergic nerve endings. Injection of toluene at doses higher than 200 mg/kg decreased ACh release and a similar decrease was suggested to occur in 8-h inhalation exposure to toluene at 1,000 ppm or higher concentrations.