A reversed-phase high-performance liquid chromatographic (RPHPLC) method has been developed for the determination of polydatin (PD) in rat plasma, bile, urine, feces and tissue homogenates using 2,3,5,4'-tetrahydroxychrysophenine-beta-d-glucoside as an internal standard. The sample pretreatment included deproteinization for plasma samples and a liquid-liquid extraction for bile, urine, feces and tissue homogenates. Separation was obtained on a C18 reversed-phase column with the mobile phase consisting of methanol and water (35:65 v/v). The flow rate was 1 ml/min and the effluent was monitored at 310 nm. The method showed good linearity over the concentration ranges employed for various matrices (r > 0.998). The quantification limits of PD in rat plasma, bile, urine, feces and tissue homogenates were 0.0251, 0.126, 0.025 microg/ml, 0.189 and 0.0378 microg/g, respectively. The accuracy and precision of the method were less than 12.0% for the various matrices. No interferences from endogenous substances were found. The method was successfully applied to study the pharmacokinetics of PD in rats after intravenous administration.