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Abstract: Objective To optimize the method of Pig-a mutation assay, and to explore the time-dependent and dose-response relationship of N-ethyl-N-nitrosourea (ENU). Methods Thirty rats were randomly assigned to 5 groups: treated with PBS (control group)or different doses of ENU (10, 20, 40 and 80 mg/kg) for 3 d by oral gavage. Blood samples were collected at 0 d, 15 d, 30 d, 45 d, 60 d, 75 d and 90 d. After enrichment, erythrocytes were incubated with Anti-CD59-APC and SYTO 13 nucleic acid dye solution. Mutant phenotype erythrocytes (RBCCD59-) and mutant phenotype reticulocytes (RETCD59-) were measured by flow cytometry to analyze mutant frequencies, and the RET percentage was determined as well. Results The RBCCD59- mutation frequency in 4 ENU groups were significantly increased in a dose- and time-dependent manner. The RETCD59- mutation frequency increased to a stable high level with a slight fluctuation,and decreased at 45 d , with the peak values observed at 30 d. The RETCD59- mutation frequency showed a dose-dependent trend in 4 ENU groups. The RET percentage in all 5 groups declined at 30 d, to a stable low level thereafter, but the trends showed no significant differences by time or group. Conclusion The optimized in vivo Pig-a mutation assay could detecte the mutagen, such as ENU, induces mutation in RBC in a time- and dose-dependent manner.