A high-performance liquid chromatographic method for the determination of berberine in plasma, urine and bile samples is described. Plasma samples were pretreated by protein precipitation with acetonitrile and urine and bile samples were pretreated by organic solvent extraction using 5% 2-propanol in methylene chloride. Berberine was determined in all samples using an octyl reversed-phase column with a mobile phase of 60-63% acetonitrile in 0.1% phosphoric acid (pH 6.0) and with UV detection at 267 nm. The detection limits for berberine in plasma, urine and bile were 18.1 ng/ml, 2.3 ng/ml and 90.4 ng/ml, respectively. The recoveries of berberine by simple deproteinization of plasma and by solvent extraction of urine were 78.3 and 82.9%, respectively. The intra-day and inter-day accuracy and precision for plasma reported as coefficients of variation and relative errors were both less then 6%. The applicability of the assay to pharmacokinetic and bioavailability studies was demonstrated by the determination of berberine in plasma, urine and bile after intravenous and intramuscular administration to rabbits at a dose of 2 mg/kg.