Highly efficient cell synchronization and metaphase chromosome accumulation in rice root tip cells were achieved. Flow cytometric analysis was performed for obtaining optimal parameters to synchronize the cell cycles. High mitotic indices (about 57.6% in root tip meristemic area) were obtained by treating seedlings with 0.5 cm length using 0.5 mM hydroxyurea at 30°C for 4 h, incubating in a hydroxyurea-free solution for 30 min, and then treating with 0.3 μM trifluralin for 3 h. After trifluralin treatment, incubation in distilled water for 15 min reduced chromosome clumping on metaphase spread. Uniformity of seed germination at the time of treatment is a critical parameter for obtaining high metaphase index. I solated rice chromosomes were suitable for flow cytometric analysis and chromosome sorting. The morphology of flow sorted metaphase chromosomes was intact.