A highly sensitive and specific atmospheric pressure chemical ionization liquid chromatography-tandem mass spectrometry method was developed for serum pharmacokinetic studies of puerarin in rats. Chromatography was carried out on a reversed-phase Phenomenex Synergi 4 μm Fusion-RP80 column (150 × 2.0 mm i.d.) using a mobile phase consisting of acetonitrile-water (10:90, v/v) in 10 mM NH4OAc with a flow rate of 0.2 mL/min. Puerarin was analyzed in the multiple reaction monitoring mode with a precursor/product ion transition of m/z 415/267. The method was demonstrated to be specific and sensitive, and a linear response was observed over a range of 2-5000 ng/mL in rat serum. The validated method was successfully applied to the characterization of the pharmacokinetics of puerarin in rat serum after oral administration to spontaneously hypertensive rats. The blood concentration-time profile of puerarin showed a rapid initial increase, reaching a maximum and then declining within 1 h. Puerarin could not be detected after 24 h. The main pharmacokinetic parameters for puerarin after oral administration were as follows: Cmax (3.54 ± 2.03 mg/L), Tmax (0.68 ± 0.37 h), AUC0-t (7.29 ± 3.79mg h/L), AUC0-∞ (9.17 ± 4.87 mg h/L), T1/2 (1.7 ± 0.6 h), CL/F (7.24 ± 4.27 L/h/kg) and V/F (17.88 ± 13.55 L/h/kg). Copyright © 2007 John Wiley & Sons, Ltd.