Semustine　(Me-CCNU)　is　an　important　alkylating　agent　used　in　the　clinical　treatment　of　cancer.　The　anticancer　mechanism　of　Me-CCNU　is　associated　with　the　formation　of　DNA　interstrand　crosslinks　(ICLs).　In　this　study,　the　ICLs　induced　by　Me-CCNU　were　investigated　using　fluorescent　assay　and　high　performance　liquid　chromatography-electronspray　ionization-tandem　mass　spectrometry　(HPLC-ESI-MS/MS).　The　results　showed　that　the　maximum　of　the　crosslinking　factor　(CF)　obtained　from　fluorescent　assay　was　8.　81%,　11.　17%,　17.　61%　and　23.　39%,　respectively,　with　the　concentration　of　Me-CCNU　of　2,　4,　8　and　16　mmol/L;　the　maximum　of　the　CF　obtained　from　HPLC-ESI-MS/MS　was　138,　256,　378　and　433　crosslinked　dG-dCs　per　10(7)　base　pairs,　respectively,　with　the　concentration　of　Me-CCNU　of　2,　4,　8　and　16　mmol/L.　By　comparing　these　two　methods　for　the　determination　of　DNA　interstrand　crosslinks,　the　CF　obtained　from　fluorescent　assay　characterized　the　crosslinks　of　the　DNA　double　strands;　on　the　other　hand,　the　CF　obtained　from　HPLC-ESI-MS/MS　characterized　the　content　of　the　crosslinked　dG-dC.　The　latter　gave　a　more　essential　quantification　of　the　ICLs　than　the　former.　An　＂induction　phase＂　was　found　by　both　of　the　two　methods,　in　which　the　CF　increased　slowly　during　the　beginning　period　of　the　reaction.　This　result　provided　evidences　for　the　clarifing　of　the　mechanism　of　the　formation　of　ICLs.