Abstract: We investigated the effect of different Cu²⁺ concentrations in the root medium on root growth, reactive oxygen species (ROS) accumulation, antioxidative response, DNA damage, plasma membrane integrity and cell viability in roots of Arabidopsis thaliana, and the toxicity range was explored with split-root technique. Primary root length was significantly inhibited. Cu²⁺ enhanced the activities of antioxidant enzymes, induced ROS accumulation and DNA damage, and reduced plasma membrane integrity. Cell death was significantly increased with increased concentration of Cu²⁺. ROS accumulation was significantly and positively correlated with the activities of total superoxide dismutase, catalase, peroxidase and ascorbate peroxidase. ROS accumulation increased linearly with DNA damage, plasma membrane integrity and cell viability. In the split-root experiment, root growth was inhibited in culture medium supplemented with 75 μmol·L^–1 Cu²⁺; however, roots were not inhibited in medium without supplementary Cu²⁺. ROS accumulation and cell death took place in only half the dish, so Cu²⁺ toxicity was local, not systemic. Local ROS accumulation in roots likely mediates the cell death caused by Cu²⁺ stress.