A rapid and simple high-performance liquid chromatographic method for determination of berberine in rat thalamus was described in this study. Thalamus samples were pretreated by protein precipitation with methanol and acetonitrile. Berberine was determined using a Hypersil C(18) column with an isocratic mobile phase of acetonitrile--0.05 M potassium dihydrogen phosphate (containing 0.5% triethylamine, pH 3.0) (30:70 v/v) and with UV detection at 265 nm. The lower limit of quantification for berberine in thalamus was 24 ng/ml, and the lowest concentration of berberine determined in rat thalamus samples was 47.5 ng/ml at 48 hours. The calibration curve for berberine was linear (r(2) = 0.9994) over the concentration range 24-6000 ng/ml. At this concentration range, the overall recoveries (91.20%-93.24%) for berberine were determined and the accuracy of the intra- and inter-day assays from rat thalamus were less than 6% RSD. Following intravenous administration of 10.2 mg/kg of Coptidis Rhizoma (CR) extract containing 3 mg/kg berberine into rats, the thalamus level of berberine increased rapidly (t(1/2alpha) = 1.93 hours), peaked at 2.48 hours with a concentration of 271 ng/g, and had a slow elimination rate (t(1/2beta) = 14.6 hours), which suggested that berberine might directly act on certain regions of the thalamus, have pharmacological effects on some cerebral dysfunctions, and be an active ingredient of huang lian jie du tang for the treatment of cerebral disease.