Dicumarol is routinely added to the 3-[4,4-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay to study the role of NAD(P)H:quinone oxido-reductase in drug activation and detoxification. We assessed the direct impact of dicumarol (a mitochondrial uncoupler) on the MTT assay. Mouse mammary tumor (EMT6) and Chinese hamster ovary (CHO) cells were treated with media containing either 10 or 1% fetal bovine serum and dicumarol (0-1000 microM) mimicking standard assay conditions. MTT, clonogenic, total reactive oxygen species (ROS), and oxygen consumption assays were performed. Significant increases in the apparent viability of EMT6 and CHO cells were observed with MTT assays after short time periods with maximum effects at 2 hr. Reduced serum concentrations intensified this effect. Conversely, significant decreases in viability for both cell lines occurred after longer incubations and serum withdrawal enhanced this effect in both cell lines. Clonogenic assays provided contrasting results where viability increased significantly only in EMT6 cells (not CHO) and was smaller than that reported by MTT. Furthermore, greater dicumarol toxicity was observed in clonogenic assays. Significant toxicity compared to control occurred after 4-hr treatment (vs. 12 hr MTT) and serum withdrawal also increased the toxicity of dicumarol with extended culture. ROS production in EMT6 and CHO cells increased in a concentration-dependent manner with 20-min dicumarol administration and thereafter declined. The EC(50) for dicumarol-induced oxygen consumption was 0.84 microM in CHO compared to 1.18 microM in EMT6 cells. Cell lines are differentially sensitive to the toxicity of dicumarol and cell survival data may be skewed by its inclusion, probably due to ROS production and mitochondrial uncoupling. Dicumarol is not recommended for inclusion in the MTT assay.