Abstract Disulfiram is used in the treatment of alcoholism. It has also shown promise as an anticancer drug. The disulfide bond in disulfiram is metabolized in vivo to diethyldithiocarbamate (DDTC) which forms an extremely strong complex with copper(II) through which it may exert its cytotoxicity. Both disulfiram and diethyldithiocarbamate inhibited K562 cell growth in the low nanomolar range. The addition of the copper(II) chelators Trien and ammonium tetrathiomolybdate to the cell culture medium significantly reduced the cytotoxicity of disulfiram and diethyldithiocarbamate towards K562 cells, which suggests that their cytotoxicity is mediated by uptake of trace amounts of copper from the culture medium. Disulfiram, but not DDTC, strongly inhibited the decatenation activity of topoisomerase IIα in the low micromolar concentration range. Disulfiram, but not DDTC, also was shown to be a topoisomerase IIα poison as evidenced by its ability to induce formation of linear DNA. However, the K/VP.5 cell line, a K562-derived etoposide-resistant cell line with reduced levels of topoisomerase II, was not cross-resistant to either disulfiram or DDTC. K562 cells depleted in GSH levels by treatment with buthionine sulfoximine did not show increased sensitivity to disulfiram or DDTC. Disulfiram and DDTC also did not increase dichlorofluorescein fluorescence in K562 cells. These latter two results suggest that copper-mediated oxidative stress may not be a significant factor in their cytotoxicity. In summary, these results showed that while disulfiram and DDTC inhibited topoisomerase IIα, this inhibition may be only partly responsible for their cytotoxicity. Support: CIHR, a Canada Research Chair in Drug Development to B.B.H., and a National Institutes of Health grant CA090787 to J.C.Y. Citation Format: Xing Wu, Jack C. Yalowich, Daywin Patel, Brian B. Hasinoff. Disulfiram is a potent inhibitor of topoisomerase II. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 830. doi:10.1158/1538-7445.AM2014-830