Multicell spheroids were exposed to DNA-damaging agents with the aim of determining whether prompt DNA damage could be predictive for cell killing and drug resistance. Chinese hamster V79 cells, SiHa human cervical carcinoma cells, and WiDr human colon carcinoma cells were grown as spheroids and exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline-1-oxide (4NQO), doxorubicin, etoposide, actinomycin D, 1-(2-nitro-1-imidazolyl)-3-aziridino-2-propanol (RSU 1069), 3-amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine), and nitrogen mustard. Average DNA damage measured using the alkali comet assay generally correlated with cell killing irrespective of exposure times or drug concentration. However, better predictive power was achieved by using DNA damage levels in individual cells to identify the fraction of cells containing sufficient numbers of DNA strand breaks to cause death. Using this concept of a "threshold" for DNA damage, cell survival could be predicted for exposure to 4NQO, tirapazamine, nitrogen mustard, RSU 1069, and actinomycin D and was largely independent of cell type. The threshold value varied for each drug. For 4NQO, tirapazamine, and RSU 1069, DNA damage equivalent to about 10,000 strand breaks/cell was not toxic to cells of any spheroid type. Conversely, for actinomycin D, any DNA damage above background levels (approximately 100 breaks) was toxic for all three cell types. For some DNA-damaging drugs, the lack of correlation between DNA damage and cell killing was also informative. For etoposide and doxorubicin, no common threshold for cell killing could be determined, consistent with the hypothesis that DNA damage is only one of the actions of these drugs leading to cell death. For MNNG, the tail moment threshold varied significantly for the different spheroid types, probably indicating differences in repair. Overall, for five of the eight drugs, DNA damage measured using the comet assay was an effective and quantitative method of predicting drug cytotoxicity in complex multicelled systems.