The present study utilized HPLC and LC‐MS approaches to investigate the pharmacokinetics and metabolism of neferine (a bisbenzylisoquinoline alkaloid). The plasma concentration‐time curves of neferine (10, 20 and 50 mg/kg, i.g.) showed double absorption peaks with the first peak at 10 min and the second peak at 1 h. The t was 15.6 h, 22.9 h and 35.5 h, for each of these doses, respectively. Neferine distributed rapidly into different organ systems, with the highest concentrations found in the liver, followed by the lung, kidney and heart at doses of 10 or 20 mg/kg. At 50 mg/kg dose, concentrations of the kidney and lung were higher than those of others. Moreover, this compound was mainly metabolized in the liver and converted partially by CYP2D6 to liensinine, isoliensinine, desmethyl‐liensinine and desmethyl‐isoliensinine. Copyright © 2007 John Wiley & Sons, Ltd.