The aim of this study is to characterize the genotoxicity of depleted uranium (DU) in Chinese Hamster Ovary cells (CHO) with mutations in various DNA repair pathways. CHO cells were exposed to 0-300 μM of soluble DU as uranyl acetate (UA) for 0-48 h. Intracellular UA concentrations were measured via inductively coupled mass spectrometry (ICP-MS) and visualized by transmission electron microscopy (TEM). Cytotoxicity was assessed in vitro by clonogenic survival assay. DNA damage response was assessed via Fast Micromethod® to determine UA-induced DNA single strand breaks. Results indicate that UA is entering the CHO cells, with the highest concentration localizing in the nucleus. Clonogenic assays show that UA is cytotoxic in each cell line with the greatest cytotoxicity in the base excision repair deficient EM9 cells and the nuclear excision repair deficient UV5 cells compared to the non-homologous end joining deficient V3.3 cells and the parental AA8 cells after 48 h. This indicates that UA is producing single strand breaks and forming UA-DNA adducts rather than double strand breaks in CHO cells. Fast Micromethod® results indicate an increased amount of single strand breaks in the EM9 cells after 48 h UA exposure compared to the V3.3 and AA8 cells. These results indicate that DU induces DNA damage via strand breaks and uranium-DNA adducts in treated cells. These results suggest that: (1) DU is genotoxic in CHO cells, and (2) DU is inducing single strand breaks rather than double strand breaks in vitro.