Phosphorylation of histone H2AX on serine 139 (gammaH2AX) occurs at sites flanking DNA double-strand breaks and can provide a measure of both number and location of these breaks within the nucleus. Because double-strand breaks are often lethal and are produced by several chemotherapeutic agents, we examined the possibility that expression of gammaH2AX after treatment might be useful as a surrogate indicator of clonogenic cell kill. Chinese hamster V79 cells were exposed for 30 min to drugs known to produce DNA double-stand breaks with different efficiencies: bleomycin, tirapazamine, doxorubicin, etoposide, 4-nitro-quinoline-N-oxide, and hydrogen peroxide. Cells were then allowed 1 h to develop foci before fixation or were plated to measure colony formation ability. Anti-gammaH2AX antibody staining was measured using flow cytometry. Flow histograms were analyzed for the percentage of cells that showed gammaH2AX levels greater than untreated cells, and this percentage was compared with the clonogenic surviving fraction. H2AX expression measured 1 h after treatment predicted cell killing for all of the drugs examined over two logs of cell kill. Moreover, predictive ability was largely independent of drug type in this cell line, and gammaH2AX levels five times background resulted in 50-90% cell kill. This method seems to provide a useful indicator of clonogenic response to treatment with selected chemotherapeutic drugs.