Chloroethylnitrosoureas　(CENUs)　are　an　important　type　of　alkylating　agent　employed　in　the　clinical　treatment　of　cancer,　However,　the　anticancer　efficacy　of　CENUs　is　greatly　decreased　by　a　DNA　repairing　enzyme,　O-6-1　alkylguanine-DNA　alkyltransferase　(AGT),　by　preventing　the　formation　of　interstrand　cross-links　(ICLs).　In　this　study,　a　combi-nitrosourea　prodrug,　namely,　N-(2-chloroethyl)-N＇-2(O-6-benzyl-9-guanine)ethyl-N-nitrosourea　(BGCNU),　which　possesses　an　O-6-benzylguanine　(O-6-BG)　derivative　and　CENU　pharmacophores　simultaneously,　was　synthesized　and　evaluated　for　its　ability　to　induce　ICLs.　The　target　compound　is　markedly　more　cytotoxic　in　human　glioma　cells　than　the　clinically　used　CENU　chemotherapies　ACNU,　BCNU,　and　their　respective　combinations　with　O-6-BG.　In　the　AGT-proficient　cells,　significantly　higher　levels　of　DNA　ICLs　were　observed　in　the　groups　treated　by　BGCNU　than　those　by　ACNU　and　BCNU,　which　indicated　that　the　activity　of　AGT　was　effectively　inhibited　by　the　O-6-BG　derivatives　released　from　BGCNU.