The concentration of serotonin (5-HT) in rat brain microdialysate before and after i. p. administration of fluoxetine hydrochloride was determined through precolumn derivatization by reversed-phase high performance liquid chromatography (RP-HPLC). A baseline separation of serotonin was achieved on a Hypersil C18 column (250 mm x 2. 0 mm, 5 microm) with acetonitrile-20 mmol/L sodium acetic solution (pH 5. 0) (45: 55, v/v) containing 20 mmol/L of sodium octanesulfonate as the mobile phase. Fluorescence detection with the excitation wavelength at 330 nm and emission wavelength at 455 nm was used for serotonin. There was good linear relationship between the peak area and concentration in the range of 0. 25 - 5. 0 nmol/L( r =0. 999 1). The limit of quantitation was 0. 25 nmol/L. The method is simple, accurate and can be applied to the determination of serotonin in biological specimen.