Chloroethylnitrosoureas　(CENUs)　are　an　important　family　of　alkylating　agents　employed　in　the　clinical　treatments　of　cancer.　They　exert　cytotoxicity　by　inducing　DNA　interstrand　crosslinks　(ICLs)　between　guanine　and　the　complimentary　cytosine,　namely　dG-dC　crosslink.　Many　investigations　have　been　performed　on　the　DNA　ICLs　involved　in　the　anticancer　efficacy　of　CENUs,　but　no　conclusive　comparisons　between　these　agents　have　been　published.　In　this　work,　the　levels　of　dG-dC　crosslink　in　calf　thymus　DNA　induced　by　four　CENUs,　including　nimustine　(ACNU),　carmustine　(BCNU),　lomustine　(CCNU)　and　fotemustine　(FTMS),　were　quantitatively　determined　using　HPLC-ESI-MS/MS.　The　obtained　time-courses　for　the　dG-dC　crosslinking　levels　indicated　that　there　is　an　induction　period　with　very　low　crosslinking　activity　at　the　initial　stage　of　the　treatment　by　BCNU　and　CCNU.　The　induction　period　provides　a　convincing　evidence　for　the　presumed　mechanism　that　the　formation　of　dG-dC　crosslinks　was　initiated　by　the　monoalkylation　of　guanine　followed　by　the　second　alkylation　of　the　complimentary　cytosine.　The　crosslinking　activity　of　ACNU　is　remarkably　higher　than　those　of　BCNU,　CCNU　and　FTMS　at　all　time　points.　The　crosslinking　activities　of　CENUs　were　found　to　be　related　to　their　stability　in　aqueous　solution.　ACNU　has　the　shortest　half-life　among　the　four　CENUs,　but　has　highest　crosslinking　levels;　on　the　contrary,　CCNU　has　the　lowest　crosslinking　activity　with　the　longest　half-life.　Moreover,　a　correlation　was　found　between　the　crosslinking　activity　and　the　anticancer　efficiency.　ACNU　with　the　highest　crosslinking　activity　showed　the　better　survival　gain　for　high-grade　glioma　than　BCNU,　CCNU　and　FTMS　as　reported　in　an　epidemiological　study.　This　suggests　that　dG-dC　crosslink　can　possibly　be　employed　as　a　potential　biomarker　for　evaluating　the　anticancer　efficiency　of　novel　CENU　drugs.　(C)　2014　Elsevier　B.V.　All　rights　reserved.