Chloroethylnitrosoureas　(CENUs),　which　are　bifunctional　alkylating　agents　widely　used　in　the　clinical　treatment　of　cancer,　exert　anticancer　activity　by　inducing　crosslink　within　a　guanine-cytosine　DNA　base　pair.　However,　the　formation　of　dG-dC　crosslinks　can　be　prevented　by　O-6-alkylguanine-DNA　alkyltransferase　(AGT),　ultimately　leading　to　drug　resistance.　Therefore,　the　level　of　AGT　expression　is　related　to　the　formation　of　dG-dC　crosslinks　and　the　sensitivity　of　cells　to　CENUs.　In　this　work,　we　determined　the　CENU-induced　dG-dC　crosslink　in　mouse　L1210　leukemia　cells　and　in　human　glioblastoma　cells　(SF-763,　SF-767　and　SF-126)　containing　different　levels　of　AGT　using　high-performance　liquid　chromatography　coupled　with　electrospray　ionization　tandem　mass　spectrometry.　The　results　indicate　that　nimustine　(ACNU)　induced　more　dG-dC　crosslinks　in　L1210　leukemia　cells　than　those　induced　by　carmustine　(BCNU),　lomustine　(CCNU)　and　fotemustine　(FTMS).　This　result　was　consistent　with　a　previously　reported　cohort　study,　which　demonstrated　that　ACNU　had　a　better　survival　gain　than　BCNU,　CCNU　and　FTMS　for　patients　with　high-grade　glioma.　Moreover,　we　compared　the　crosslinking　levels　and　the　cytotoxicity　in　SF-763,　SF-767　and　SF-126　cells　with　different　AGT　expression　levels　after　exposure　to　ACNU.　The　levels　of　dG-dC　crosslink　in　SF-126　cells　(low　AGT　expression)　were　significantly　higher　than　those　in　SF-767　(medium　AGT　expression)　and　SF-763　(high　AGT　expression)　cells　at　each　time　point.　Correspondingly,　the　cytotoxicity　of　SF-126　was　the　highest　followed　by　SF-767　and　SF-763.　The　results　obtained　in　this　work　provided　unequivocal　evidence　for　drug　resistance　to　CENUs　induced　by　AGT-mediated　repair　of　DNA　ICLs.　We　postulate　that　the　level　of　dG-dC　crosslink　has　the　potential　to　be　employed　as　a　biomarker　for　estimating　drug　resistance　and　anticancer　efficiencies　of　novel　CENU　chemotherapies.