Radioactive copper (II) (diacetyl-bis N4-methylthiosemicarbazone) (Cu-ATSM) isotopes were originally developed for the imaging of hypoxia in tumors. Because the decay of a sup.64 Cu atom is emitting not only positrons but also Auger electrons, this radionuclide has great potential as a theranostic agent. However, the success of sup.64 Cu-ATSM internal radiation therapy would depend on the contribution of Auger electrons to tumor cell killing. Therefore, we designed a cell culture system to define the contributions to cell death from Auger electrons to support or refute our hypothesis that the majority of cell death from sup.64 Cu-ATSM is a result of high-LET Auger electrons and not positrons or other low-LET radiation. Chinese hamster ovary (CHO) wild type and DNA repair-deficient xrs5 cells were exposed to sup.64 Cu-ATSM during hypoxic conditions. Surviving fractions were compared with those surviving gamma-radiation, low-LET hadron radiation, and high-LET heavy ion exposure. The ratio of the D sub.10 values (doses required to achieve 10% cell survival) between CHO wild type and xrs5 cells suggested that sup.64 Cu-ATSM toxicity is similar to that of high-LET Carbon ion radiation (70 keV/mum). gammaH2AX foci assays confirmed DNA double-strand breaks and cluster damage by high-LET Auger electrons from sup.64 Cu decay, and complex types of chromosomal aberrations typical of high-LET radiation were observed after sup.64 Cu-ATSM exposure. The majority of cell death was caused by high-LET radiation. This work provides strong evidence that sup.64 Cu-ATSM damages DNA via high-LET Auger electrons, supporting further study and consideration of sup.64 Cu-ATSM as a cancer treatment modality for hypoxic tumors. Keywords: auger electron, high LET, sup.64 Cu-ATSM , DNA double strand break, CHO