Particulate hexavalent chromium ((Cr(VI)) compounds are human lung carcinogens. These compounds induce DNA damage, chromosome aberrations, and concentration-dependent cell death in human and Chinese hamster ovary (CHO) cells. The relationship between Cr(VI)-induced DNA damage and chromosome aberrations is poorly understood. Accordingly, this study focused on examining the role of Ku80, a gene involved in nonhomologous end-joining repair, in particulate chromate-induced cytotoxicity and chromosome damage in CHO cells. Three different cell lines were used: CHO-K1 (parental), xrs-6 (Ku80 deficient), and 2E (xrs-6 complemented with Ku80 gene). Levels of cell death were higher in xrs-6 cells when compared to wild type, suggesting that Ku80 was important for protecting cells from lead chromate. However, Ku80 played no role in protecting cells from particulate Cr(VI)-induced chromosome instability (CIN) as gene complementation with Ku80 (2E cells) studies and uptake experiments showed similar frequency and amounts of chromosome damage between the cell lines and that any observed difference based on administered concentration was actually due to differences in Cr(VI) uptake. The spectrum of chromosome damage was also unaffected by Ku80 deficiency. These data indicate that Ku80 protects cells from cytotoxicity but is not involved in protecting cells from particulate chromate-induced CIN.