Semustine　(Me-CCNU,　1-(2-chloroethyl)-3-(4-transmethylcyclohexyl)-1-　nitrosourea)　is　significant　anticancer　agents　in　the　clinical　treatment　of　human　malignancies.　Several　lines　of　evidences　indicate　that　its　cytotoxic　activity　is　related　to　the　interstrand　cross-linking　of　DNA.　In　this　study,　the　agarose　gel　electrophoresis　and　fluorometric　assay　were　carried　out　to　detect　the　NDA　interstrand　cross-linking　caused　by　semustine.　It　was　found　that　the　cross-links　of　DNA　induced　by　drugs　increased　progressively　with　the　reaction　time,　tending　towards　a　maximum　stable　value　at　about　8h.　Simultaneously,　a　buffering　stage　in　the　beginning　of　DNA　cross-linking　was　apparent.　The　results　suggest　that　the　formation　of　DNA　interstrand　cross-linking　may　undergo　an　induction　period.　It　is　proposed　that　the　generation　of　DNA　interstrand　cross-linking　go　through　two　steps　and　is　affected　by　the　concentration　of　drugs.　©　2008　IEEE.