Abstract: 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.