Abstract: To study the tissue distribution and excretion of indomethacin 5-fluorouracil-1-ylmethyl ester (IFM) metabolite 5-fluorouracil in rats, an accurate and specific high performance liquid chromatography method for quantifying IFM in rat plasma and tissues was developed. Biological samples were prepared by liquid-liquid extraction and separated on a Diamonsil^TM C₁₈ column (250 mm×4.6 mm ID, 5 μm). The mobile phase for tissue samples, plasma samples and feces samples were composed of methanol-water-36% acetic acid (3∶96.9∶0.1, v/v) and the mobile phase for urine samples was a mixture of methanol-water-36% acetic acid (10∶89.9∶0.1, v/v). The eluate was monitored by UV absorbance at 260 nm. After a single ig dose of 100 mg·kg^-1 IFM in rats, 5-Fu was mainly distributed in stomach, small intestine, and liver. The concentrations of 5-fluorouracil in other tissues and plasma were low. The excretion of 5-Fu in urine and feces amounted to 0.006 5% and 0.063% of the dose, respectively. The method is shown to be accurate and specific, and suitable for preclinical pharmacokinetic studies of IFM.