Bifunctional　alkylating　agent　1,3-Bis-(2-chloroethyl)-1-nitrosourea　(BCNU)　is　a　significant　anticancer　drug　in　the　clinical　treatment　of　human　malignancies.　Several　lines　of　evidences　indicated　that　the　cytotoxic　activity　of　BCNU　was　related　to　their　ability　of　inducing　DNA　interstrand　cross-links　(ICLs).　In　the　present　work,　the　formation　of　the　ICLs　from　the　treatment　of　calf　thymus　DNA　with　BCNU　was　studied　with　HPLC-ESI-MS/MS　analysis　and　QM/MM　computations.　Double　strand　calf　thymus　DNA　was　treated　with　BCNU　and　digested　enzymatically　down　to　the　nucleoside　level.　The　hydrolysates　were　analyzed　by　HPLC　separation　followed　by　electrospray　ionization　tandem　mass　spectrometric　conformation.　The　main　ICL　product,　1-[N3-deoxycytidyl]-2-[N　1-deoxyguanosyl]-ethane,　was　characterized　by　selected　reaction　monitoring　(SRM)　mode　with　ion　transitions　of　m/z　521m/z　405　([M+H　+-dR]+)　and　m/z　521m/z　289　([M+H+-2dR]　+).　QM/MM　computations　were　carried　out　with　ONIOM　method　to　investigate　molecular　geometric　structure　of　the　DNA　double　helixes　containing　various　cross-links.　A　stable　structure　of　the　dC(N3)-CH　2CH2-dG(N1)　ICL　was　obtained,　which　was　consistent　with　the　result　of　HPLC-ESI-MS/MS　analysis.　©2009　IEEE.