Aristolochic acid (AA) was incubated with 2'-deoxyadenosine 5'-monophosphate (dAp) in mild acid phosphate buffer in vitro for 12 hours, using either enzymatic activation (by xanthine oxidase) or chemical activation (by zinc) to synthesize AA-DNA adducts, and the solvents were evaporated under vacuum. The AA-DNA adducts were characterized by multiple mass spectrometric techniques. Electrospray ionization/tandent mass spectrometry (ESI-MS/MS) was used for the characterization of the AA-DNA adducts. Full scan spectra were obtained lit the negative]oil mode and the quasi-molecular ion peaks of the AA-DNA adducts were m/z 621 and m/z 591 respectively. Crude extracts were analyzed by, ESI-MSn technique to investigate structural information about the fragmentation rules of AA-DNA adducts in negative mode. Using the high accuracy mass data and isotope pattern Of Super high resolution Fourier transform-ion cyclotron resonance mass spectrometry ( FT-ICRMS), the AA-DNA adducts were identified further. The results indicated that AA-DNA adducts were synthesized lit vitro This method is a powerful tool for detection and identification of AA-DNA adducts. Compared with P-32-postlahelllng analysis this method has the advantages of simple operation, rapid measurement and accurate determination.