To develop and verify a flow cytometric measurement of reticulocytes (RETs) micronucleus in rat bone marrow.In our flow cytometric protocol, reticulocytes, leukocytes and DNA were labeled by anti-CD71-fluorescein isothiocyanate (FITC), anti-CD45-phycoerythrin (PE) and DRAQ5, respectively. Sprague-Dawley (SD) rats were assigned to four treatment groups randomly, and were exposed to ethyl methanesulfonate (EMS), cyclophosphamide (CP), ethyl nitrosourea (ENU) and colchicine (COL) respectively. Each treatment group was divided into four subgroups (5 rats per subgroup) according to different exposure dosage. A exposure dose of 0 was used as vehicle control for each group. Rats were administered with testing mutagens by gavage twice with a 24 h interval. Bone marrow from both femurs were collected 24 h after the last administration. The frequency of micronucleated reticulocytes (MN-RETs) and the percentage of reticulocytes (RETs%) were determined by flow cytometric measurement established in this study. And the manual counting method with microscope (by Giemsa staining) was conducted at the same time.A method for detection of reticulocyte micronucleus in bone marrow based on flow cytometry was successfully established. The MN-RETs in rat bone marrow of 20 SD rats treated by vehicle (i.e., background value of MN-RETs) was 0.83‰±0.12‰ by this method. The background value of MN-RETs in manual enumeration method was 1.43‰±0.44‰. It was obvious that the flow cytometric method had lower background value and more stable results. The trend, in which MN-RETs ascended and RETs% descended with increasing dose, can be detected by both methods in rats that exposed to EMS, CP, ENU and COL. Both methods were good to detect the correlation of induced-MN-RETs with four testing mutagens (the correlation coefficients were ranged from 0.834 3 to 0.913 7).With its sensitivity, rapidity, easy operation and low background value, the three-color flow cytometric enumerative protocol established in our laboratory can be used as a good substitute for manual micronucleus counting method and used in genotoxicity assessment of chemical substances.