The mitotic shake-off method revealed the remarkable variation of radiosensitivity of HeLa cells during the cell cycle: M phase shows the greatest radiosensitivity and late S phase the greatest radioresistance. This method harvests all M-phase cells with a round shape, making it impossible to further subdivide M-phase cells. Recently, the fluorescent ubiquitination-based cell cycle indicator (Fucci) was developed; this system basically causes cells in G₁ to emit red fluorescence and other cells to emit green fluorescence. Because the green fluorescence rapidly disappears at late M phase, two-dimensional flow cytometry analysis can usually detect a green^high/red^low fraction including S-, G₂- and early M-phase cells but not a transitional fraction between green^high/red^low and green^low/red^low including late M-phase cells. However, combining the shake-off method concentrated the transitional fraction, which enabled us to separate early and late M-phase cells without using any drugs. Here we demonstrate for the first time that cells in early M phase are more radiosensitive than those in late M phase, implying that early M phase is the most radiosensitive sub-phase during the cell cycle.