Toxic　effects　of　alkylating　agents　on　cell　survival　and　DNA　damage　was　investigated　on　a　single　cell　level.　In　this　study,　single　cell　gel　electrophoresis　assay　(SCGE)　was　used　to　detect　and　compare　DNA　damages　induced　by　monofunctional　and　bifunctional　alkylating　agents　under　different　concentrations.　Linear　positive　correlation　between　drug　concentration　and　DNA　damages　has　been　demonstrated.　To　further　investigate　the　possible　induction　of　DNA　crosslinking　by　ACNU,　DNA　cleavage　reagent　was　used,　and　results　show　that　ACNU　can　cause　significant　DNA　crosslinks.　The　result　have　provided　theoretical　basis　for　clarifying　carcinogenic　and　anti-cancer　mechanisms　of　alkylating　agents.　©,　2015,　Beijing　Institute　of　Technology.　All　right　reserved.