To test the hypothesis that attenuation in G2-phase checkpoint control leads to elevated frequencies of unrejoined chromosome breaks in mitosis, the relationship between G2-phase cell cycle checkpoint control and unrejoined chromosome break frequencies after radiation exposure was examined in cells of 10 human tumor cell lines: 8 squamous cell carcinoma cell lines and 2 lymphoblastoid cell lines. Most of the delay in progression through the cell cycle seen in the first cell cycle after radiation exposure in these cell lines was due to blocks in G2 phase, and there were large cell line-dependent variations in the length of the G2-phase block. There was a highly significant inverse correlation between the length of G2-phase delay after radiation exposure and the frequency of induced unrejoined chromosome breaks seen as chromosome terminal deletions in mitosis. This observation supports the hypothesis that the signal for G2-phase delay in mammalian cells is an unrejoined chromosome break and that attenuation of G2-phase checkpoint control allows cells with unrejoined breaks to progress into mitosis. Attenuation in G2-phase checkpoint control was not associated with alterations in the frequency of induced chromosome rearrangements, suggesting that most chromosome rearrangements develop prior to G2 phase, and there was no significant relationship between the length of G2-phase delay and inherent radiation sensitivity, suggesting that unrejoined chromosome breaks are not the primary toxic lesion induced by radiation in mammalian cells.