OBJECTIVE： To screen the dosing period of positive drug cyclophosphamide （CP） and ethyl methanesulfonate （EMS） in alkaline comet assay of rats in vivo. METHODS： SD rats were randomly divided into blank control group， CP group （40 mg/kg， i.p.） and EMS group （100 mg/kg， i.g.）， 9 rats in each group， every 24 h， for consecutive 4 times. 3 h after secondary， third and forth medication （27， 51， 75 h after first medication）， 3 rats were randomly selected and sacrificed in each group. Liver and stomach samples were collected. After single cell suspension preparation， production， lysis， chain breaking， electrophoresis and dyeing， the evaluation indicators， including tail DNA％， tail length （TL） and tail moment （TM）， Comet Assay Ⅳ comet assay data analysis system was used to screen the optimal dosing period. RESULTS： There was no statistical significance in Tail DNA％， TL and TM of liver and stomach in blank control group at 3 test time points （P＞0.05）， indicating that the comet assay system was stable. Tail DNA％， TL （except for stomach at 27 h） and TM of liver and stomach in CP and EMS groups were increased significantly， compared with blank control group （P＜0.05）. There was no statistical significance in Tail DNA％， TL and TM of liver in CP group at the three test time points （P＞0.05）， tail DNA％ and TL of stomach in CP group were maximal at 51 h. TM was increased with dosing period. Tail DNA％ of liver in EMS group， TM of stomach and liver were increased with dosing period. Tail DNA％ of the stomach， TL of liver and stomach were the highest at 51 h in EMS group. CONCLUSIONS： During in vivo alkaline comet assay， it is recommended that CP and EMS be administered once every 24 h and three times continuously.