A high performance liquid chromatographic pre-column derivatization method with fluorescence detection for the analysis of glutamine (Gln) in rat intestinal mucosa is presented. Gln was derivatized with o-phthalaldehyde and 3-mercaptopropionic acid under an alkaline condition and separated by reversed-phase liquid chromatography on a Lichrosorb RP18 column (150 mm x 4.6 mm i.d., 5 microm). The mobile phase was consisted of 50 mmol/L phosphate buffer (pH 7.0)-acetonitrile (94:6, V/V) with a flow rate of 2.0 mL/min. The excited and emitted wavelength were selected at 230 nm and 389 nm respectively. The volume ratio of samples and derivatization reagent solution was 4:1 (V/V). The detection limit of Gln was 25 micromol/L (S/N= 3.5) and the regression equation was A = 16.9405C + 179.9339, r = 0.9996 at the linear range of 50-3200 micromol/L. The day-to-day deviation was 6.97% (n = 3) and the retention time of Gln was 3.158 min. This method is rapid, simple and highly sensitive, and has been applied to the determination of Gln in intestinal mucosa.