Nitidine chloride (NC) is a benzophenanthridine alkaloid isolated from the roots of (Roxb.) DC, a widely used traditional herbal medicine. Several reports have revealed NC's multiple pharmacologic properties. The inhibitory effects of NC on human cytochrome P450 enzymes were investigated in the present study. We found that NC caused time- and concentration-dependent inhibition of CYP2D6, and more than 50% of CYP2D6 activity was suppressed after a 15-minute incubation with NC at 100M in the primary incubation mixtures, with of 4.36M, of 0.052 minute, and a partition ratio of approximately 290. Moreover, the loss of CYP2D6 activity required the presence of NADPH. Superoxide dismutase/catalase and glutathione showed minor protection against the NC-induced enzyme inhibition. Quinidine as a competitive inhibitor of CYP2D6 slowed down the inactivation by NC. Trapping experiments using -acetylcysteine demonstrated that quinone and/or carbene intermediate(s) were/was generated in human liver microsomal incubations with NC. In addition, potassium ferricyanide prevented the enzyme from the inactivation mediated by NC, which provided evidence that inhibition of CYP2D6 resulted from heme destruction by the formation of a carbene-iron complex. CYP1A2 was found to be the major enzyme involved in the generation of NC quinone metabolites. In conclusion, NC is a mechanism-based inactivator of CYP2D6. The generation of a carbene intermediate might be mainly responsible for the enzyme inactivation.