Acute administration of fluoxetine (1, 10 and 20 mg/kg i.p.) increased extracellular levels of serotonin (5-hydroxytryptamine, 5-HT) in the frontal cortex, ventral hippocampus and raphe nuclei as measured by in vivo microdialysis in anaesthetized rats. In the frontal cortex, fluoxetine showed a marked dose-response effect whereas in the ventral hippocampus and raphe nuclei the fluoxetine-induced effect was maximum at 10 mg/kg. However, the maximal increase in 5-HT was observed in the cell body-containing area, the raphe nuclei. The order of changes in extracellular 5-HT was raphe nuclei > ventral hippocampus > frontal cortex. Our results add further arguments in favour of the key role played by raphe nuclei in the mechanism of action of serotoninergic antidepressant drugs.