The role of metallothionein (MT) in mitigation of oxidative DNA damage (ODD) induced by either cadmium (Cd) or the direct oxidant hydrogen peroxide (H(2)O(2)) was systematically examined using MT-I/II double knockout (MT-null) or MT-competent wild-type (WT) cells. Both toxicants were much more lethal to MT-null cells (Cd LC(50) = 6.6 μM; H(2)O(2) LC(50) = 550 μM) than to WT cells (Cd LC(50) = 16.5 μM; H(2)O(2) LC(50) = 930 μM). Cd induced concentration-related MT increases in WT cells, while the basal levels were undetectable and not increased by Cd in MT-null cells. ODD, measured by the immuno-spin trapping method, was minimally induced by sub-toxic Cd levels (1 or 5 μM; 24 h) in WT cells, but markedly increased in MT-null cells (>430 %). Similarly, ODD was induced to higher levels by lower concentrations of H(2)O(2) in MT-null cells than WT cells. Transfection of MT-I into MT-null cells reduced both Cd- and H(2)O(2)-induced cytolethality and ODD. Cd increased the expression of the oxidant defense genes, HO-1, and GSTa2 to a much greater extent in MT-null cells than in WT. Cd or H(2)O(2) exposure increased the expression of key transport genes, Mrp1 and Mrp2, in WT cells but not in MT-null cells. MT protects against Cd- and H(2)O(2)-induced ODD in MT-competent cells possibly by multiple mechanisms, potentially including direct metal ion sequestration and sequestration of oxidant radicals by MT. MT-deficient cells appear to adapt to Cd primarily by turning on oxidant response systems, while MT-competent cells activate MT and transport systems.