Benzo[a]pyrene diol epoxide (BPDE) was reacted in vitro with (2'-deoxy)nucleotides and with calf thymus DNA. The modified DNA was enzymatically hydrolyzed to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase 1), nuclease P1 and snake venom phosphodiesterase (SVP). Most of the unmodified nucleotides were removed using solid phase extraction (SPE) in a polystyrene divinylbenzene copolymer. Three adducts could be detected and identified using capillary zone electrophoresis(negative)-ion electrospray ionization-mass spectrometry (CZE-(-)ESI-MS) in conjunction with sample stacking. This way, not only a BPDE-dGMP adduct [(M-H)- at m/z 648], a well-known nucleotide adduct, could be retrieved, but also a BPDE-dAMP [(M-H)- at m/z 632] and a BPDE-dCMP adduct [(M-H)- at m/z 608] could be detected for the first time. The presence of the prominent ion at m/z 195 (the deoxyribose-phosphate ion) in the three low-energy collision-activated decomposition (CAD) spectra indicated that the adducts were formed through base-alkylation. CZE-positive ion ESI-MS/MS experiments were performed to obtain further information on base-alkylation. The absence of the loss of NH3 from the nucleobase in each CAD spectrum points to an alkylated exocyclic NH2 position of the nucleobase. So, the three adducts could be identified as BPDE-N-2-dGMP BPDE-N-6-dAMP and BPDE-N-4-dCMP using CZE-ESI-MS and CZE-ESI-MS/MS.