Regulatory guidelines suggest testing chemicals up to cytotoxic doses in chromosomal-aberration assays. To investigate the utility and limitations of various cytotoxicity indicators we used Chinese hamster ovary (CHO) cells to test 8 chemicals with differing ratios of cytotoxicity to clastogenicity. We measured immediate or delayed cell killing and growth inhibition (ATP levels, cell counts, colony-forming efficiency, CFE) and cell-cycle perturbations (mitotic index, MI; average generation time, AGT). Aberrations (abs) were scored 10 and 24 h from the beginning of the 3-h treatment. All 8 compounds induced abs at concentrations that reduced cell growth at 24 h by 50% or less. Concentrations of each chemical which induced at least 15% cells with abs, gave little loss of CFE (0-20%) for mitomycin C, adriamycin, cadmium sulfate and 2,6-diaminotoluene in contrast to the marked loss of CFE (70-80%) for eugenol (EUG), 2-aminobiphenyl and 8-hydroxyquinoline (8-HQ). 2,4-Diaminotoluene (2,4-DAT) was intermediate. Higher aberration yields were found at 24 h than at 10 h, even when minimal cell-cycle delay was detected by AGT estimates from BrdUrd-labeled cells. Cells with multiple abs were seen at 24 but not at 10 h, and often confirmed clastogenicity when there was only a weak increase in the percentage of cells with aberrations. Total ATP per culture did not always correlate with cell number, especially at later times after treatment. This is likely due to metabolic perturbations or altered cell biomass that are known to affect cell ATP content. MI suppression often did not correlate with AGT, e.g., only small increases in AGT were seen for 8-HQ, 2,4-DAT and EUG despite severe mitotic suppression at 10 h. By 24 h the MI for all chemicals had recovered, sometimes exceeding control levels. Marked mitotic accumulation was seen at 10 h for 2,4-DAT, indicating cell synchrony. Thus, the MI has limited value for dose selection. In conclusion, even weakly active chemicals were detected at a single time without exceeding a 50% growth reduction at 24 h.