The coumermycin antibiotic novobiocin, which interacts with the nuclear enzyme topoisomerase II, produced supra-additive toxicity to WEHI-3B D+ leukemia cells at clinically achievable concentrations, when combined with teniposide (VM-26) or etoposide (VP-16). Simultaneous exposure of cells to both agents was required for maximum efficacy of the combination. Novobiocin also produced supra-additive toxicity to A549 human lung carcinoma cells when combined with VM-26 or VP-16. At concentrations above the peak plasma levels achievable in patients, novobiocin lost its potentiating activity. Exposure of WEHI-3B D+ cells to novobiocin did not modify the cytotoxicity produced by the topoisomerase II inhibitor m-AMSA, whereas, in contrast, novobiocin antagonized the cytotoxicity of m-AMSA in A549 cells. Although it has been suggested that inhibitors of the syntheses of DNA and RNA interfere with the cytotoxic activity of the epipodophyllotoxins, maximum potentiation of the cytotoxicities of VP-16 and VM-26 occurred at novobiocin concentrations that decreased the rates of synthesis of both DNA and RNA in WEHI-3B D+ cells by about 50%. The number of DNA-topoisomerase-II covalent complexes stabilized by VM-26 in WEHI-3B D+ cells was greatly increased when cells were exposed simultaneously to VM-26 and novobiocin for 1 hr, but not when cells were treated with m-AMSA and novobiocin for the same period of time. Novobiocin did not affect the amount of covalent complexes produced by VM-26 in isolated nuclei, suggesting that the potentiating activity of novobiocin was not due to its direct interaction with the nuclear topoisomerase II enzyme. Our findings suggest that therapeutic levels of novobiocin may be capable of enhancing the clinical activities of VP-16 and VM-26.