Aim:　To　investigated　the　mechanism　of　Chloroethylnitrosoureas　(CENUs)　leading　to　secondary　tumors　after　therapy,　three　kinds　of　CENUs,　namely,　Nimustine　(ACNU),　Carmustine　(BCNU)　and　Semustine　(MeCCNU)　were　used　to　investigated　drug-induced　DNA　interstrand　crosslinks(ICLs).　Method:　The　alkaline　comet　assay　was　adopted　to　compare　DNA　damages　induced　by　CENUs　under　different　concentrations.　Results:　With　the　increase　of　drug＇s　concentration,　DNA　migration　exhibited　a　positive　concentration-dependent　relationship　in　all　three　drugs.　ACNU　was　shown　to　cause　significant　crosslinking.　The　other　two　drugs　also　induced　crosslinking,　but　this　could　not　be　analyzed　at　high　concentrations　due　to　the　high　cytotoxicity　of　the　drugs　which　caused　cells　death.