Abstract: A sensitive and specific method was developed for the isolation of daphnetin from biological specimens and its quantitative determination by the application of silica gel TLC and UV spectrophotometry. This method was adapted for the study of the metabolic fate of daphnetin and its pharmacokinetics in rats.The pattern of decline of the plasma level of daphnetin after I V administration exhibited the characteristics of a two-compartment open model. The pharmacokinetic parameters are as follows: t_1/2α 0.043hr, t_1/2β 0.347 hr, V_c 0.372L/kg, Vd 0.653 L/kg, Kel 3.507 hr^-1, Cl 1.303 L/hr/kg.Absorption of the drug from the gastrointestinal tract was found to be fairly rapid and complete. After intravenous and oral administration, the tissue distribution and the amount excreted in urine, bile and faeces within 24 hours were observed directly in rats. These results revealed that daphnetin was distributed widely in the body and excreted mainly in urine at a faily rapid rate which was in agreement with the results of pharmacokinetic analysis. In vitro experiments provided evidence that daphnetin was slightly resolved in the gastrointestinal tract, and might be metabolized by blood and various tissue such as liver, lung and kidney. The drugplasma binding rate was found to be 14.6%.