Chloroethylnitrosoureas　(CENUs)　are　an　important　family　of　alkylating　agents　used　in　the　clinical　treatment　of　cancer.　Their　anticancer　mechanism　primarily　involves　the　formation　of　DNA　interstrand　crosslinks　(ICLs)　induced　by　the　chloroethyldiazonium　ion　derived　from　the　decomposition　of　CENUs.　In　this　work,　the　mechanism　for　the　formation　of　ICLs　was　investigated　by　density　functional　theory　(DFT)　with　B3LYP,　wB97XD,　and　M062X　functinoals　using　conductor-like　polarizable　continuum　model　solvent　model.　Three　pathways　leading　to　the　formation　of　three　types　of　GC　crosslinks　were　compared.　G(N1)C(N3)　crosslink　is　predicted　to　be　the　dominant　crosslinking　product　other　than　G(O6)C(N4)　and　G(N2)C(O2)　crosslinks,　which　is　consistent　with　the　previous　results　obtained　from　QM/MM　computations.　The　results　indicate　that　the　formation　of　the　G(N1)C(N3)　crosslink　via　pathway　A　is　the　most　favorable　mechanism　from　both　kinetic　and　thermodynamic　standpoints.　In　this　pathway,　the　chloroethyldiazonium　ion　alkylates　guanine　on　the　O6　site　followed　by　intramolecular　cyclization　to　form　O6,N1-ethanoguanine　(4).　The　cytosine　then　reacts　with　intermediate　4　on　the　C　atom　to　yield　the　G(N1)C(N3)　crosslink.　This　work　provides　reasonable　explanations　for　the　supposed　mechanism　of　CENUs-induced　ICLs　formation　obtained　from　experimental　investigations.　(c)　2012　Wiley　Periodicals,　Inc.