Chloroethylnitrosoureas　(CENUs)　are　bifunctional　alkylating　agents　widely　used　for　the　clinical　treatment　of　cancer.　They　exert　anticancer　activity　by　inducing　DNA　interstrand　cross-links　(ICLs)　within　GC　base　pairs　to　form　dG-dC　cross-links.　This　lesion　inhibits　DNA　double　strand　separation　during　replication　and　transcription　and　results　in　the　apoptosis　of　cancer　cells.　However,　O-6-alkylguanine　DNA　alkyltransferase　(AGT)　repairs　the　DNA　ICLs　by　removing　the　alkyl　group　at　the　06　position　of　either　O-6-(2-chloroethyl)deoxyguanosine　(O-6-ClEtdGuo)　or　N1,O-6-ethanodeoxyguanosine　(N1,O-6-EtdGuo),　which　are　intermediates　in　the　formation　of　dG-dC　cross-links.　The　action　of　AGT　leads　to　drug　resistance　against　CENUs.　O-6-Benzylguanine　(O-6-BG)　was　identified　as　an　effective　ACT　inhibitor　that　enhances　the　antitumor　effects　of　CENUs.　In　this　study,　the　effect　of　O-6-BG　on　the　formation　of　dG-dC　cross-links　was　investigated　by　treating　human　brain　glioma　SF767　cells　with　1-[(4-amino-2-methyl-5-pyrimidinyl]methyl]-3-(2-chloroethyl)-3-nitrosourea　(ACNU).　The　levels　of　dG-dC　cross-link　were　determined　using　stable　isotope　dilution　high-performance　liquid　chromatography　electrospray　ionization　tandem　mass,　spectrometry　(HPLC-ESI-MS/MS).　The　results　indicated　that　ACNU　induced　higher　levels　of　dG-dC　cross-link　in　SF767　cells　pretreated　with　O-6-BG　compared　to　cells　without　O-6-BG　pretreatment.　The　highest　dG-dC　cross-linking　levels　were　generally　observed　at　12　h　for　all　drug　concentration　groups,　a　result　which　was　consistent　with　cytotoxicity　assay.　These　results　provided　direct　evidence　for　the　enhancement　of　dG-dC　cross-linking　levels　caused　by　the　inhibition　of　ACT　by　O-6-BG.　These　data　indicate　that　dG-dC　crosslinks　may　be　developed　as　a　biomarker　for　evaluating　the　activity　of　novel　O-6-BG　analogues　as　AGT　inhibitors　for　combination　therapy　with　CENUs.