A sensitive and rapid method of high-performance liquid chromatography with fluorescence detection (HPLC-FLD) was developed and applied for the determination of aristolochic acid I (AAI) in rat urine and plasma. Prior to the HPLC analysis, the samples were derivatized to increase fluorescence character. The linear ranges of the calibration curves were 0.48-48ng in urine and 0.23-69ng in plasma. The intra- and inter-day precisions referred by relative standard deviation (RSD) were less than 3.2% and 4.0% for urine samples as well as less than 9.4% and 10.8% for plasma samples. The limits of quantification were 0.09 and 0.07ng in urine and plasma, respectively. The developed method was successfully applied for the determination of AAI in rat urine and plasma samples collected after the oral administrations of AAI standard and Aristolochia contorta Bge. (Madouling) or Aristolochia manshuriensis (Guanmutong) herbal extracts. The ratios of the detected AAI amount in urine compared to the dosing amount of AAI were approximately constant. The concentrations of AAI in rat plasma were much lower than those in urine. The obtained results indicated that the metabolism of the AAI standard and AAI-containing herbs might be different, probably due to the complicated and multiple components in the herbs.