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Semustine (Me-CCNU, 1-(2-chloroethyl)-3-(4-trans- methylcyclohexyl)-l-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.