We have optimized a flow cytometric DNA alkaline unwinding assay to increase the sensitivity in detecting low levels of DNA damage (strand breaks and alkali-labile sites) and to permit the measurement of the extent of DNA damage within each cell cycle compartment. The lowest γ radiation dose that induced detectable DNA damage in each cell cycle phase of HeLa and CEM cells was 10 cGy. The lowest H2O2 concentration that induced detectable DNA damage in each cell cycle phase was 0.5 μM in HeLa cells, and 1-2.5 TμM in CEM cells. For both HeLa cells and CEM cells, DNA damage in each cell cycle compartment increased approximately linearly with increasing doses of γ radiation and H2O2. Although untreated HeLa and CEM cells in S phase consistently exhibited greater DNA unwinding than did G1 or G2 cells (presumably due to DNA strand breaks associated with replication forks), there was no difference between the susceptibility of G0/G1, S and G2/M phase cells to DNA damage induced by γ radiation or H2O2, or in the rate of repair of this damage. In each cell cycle phase, the susceptibility to γ radiation-induced DNA damage was greater in CEM cells than in HeLa cells. In contrast to the lack of cell cycle phase-specific DNA damage induced by exposure to γ radiation or H2O2, the cancer chemotherapeutic drug doxorubicin (adriamycin) predominantly induced DNA damage in G2 phase cells.