Mutations in the BCR/ABL kinase domain play a major role in resistance to imatinib mesylate (IM). We report here that BCR/ABL kinase stimulates reactive oxygen species (ROS), which causes oxidative DNA damage, resulting in mutations in the kinase domain. The majority of mutations involved A/T→G/C and G/C→A/T transitions, a phenotype detected previously in patients, which encoded clinically relevant amino acid substitutions, causing IM resistance. This effect was reduced in cells expressing BCR/ABL(Y177F) mutant, which does not elevate ROS. Inhibition of ROS in leukemia cells by the antioxidants pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine (NAC), and vitamin E (VE) decreased the mutagenesis rate and frequency of IM resistance. Simultaneous administration of IM and an antioxidant exerted better antimutagenic effect than an antioxidant alone. Therefore, inhibition of ROS should diminish mutagenesis and enhance the effectiveness of IM. (Blood. 2006;108:319-327)