Previous results using a variety of methods have shown no significant difference in induction or rejoining of radiation-induced DNA single-strand breaks (SSBs) as a function of cell-cycle position. However, concurrent analysis of DNA damage and cell-cycle position measured using the alkaline comet assay indicates cycle-dependent differences in "tail moment" which has been shown to be a measure of SSBs. Unirradiated S phase cells showed a significantly higher tail moment presumably as a result of the presence of active replication sites. The time required to repair half of the damage appeared to be consistently shorter for G1 cells than for cells in other phases of the cell cycle (3.7 min versus approximately 5 min). Although early kinetics of rejoining (within 5 min) was identical for CHO, V79 and repair-deficient TK6 cells, TK6 cells subsequently repaired fewer breaks than did CHO or V79 cells. S phase TK6 cells rejoined SSBs more slowly than cells in the other phases; differences in rejoining rates could not be explained by the presence of a subset of slowly repairing TK6 cells. We conclude that the comet assay detects subtle differences in the tail moment when cells in different phases of the cell cycle are irradiated and allowed to repair damage. The biological importance of these observations remains to be determined.