Antioxidant activity is believed to be an important intracellular function of metallothioneins (MT), yet the specific mechanisms of their antioxidant action are not known. Under conditions when cells are challenged with elevated concentrations of free copper as a result of metabolic disturbances or environmental and occupational exposures, MTs may be ideally suited for antioxidant function as effective copper chelators. In the study presented here, we tested this hypothesis using a recently established model of copper nitrilotriacetate-induced oxidative stress in HL-60 cells. Since copper-induced oxidative stress triggers apoptosis, we further investigated antiapoptotic function of MTs in HL-60 cells. Using a Sephadex G-75 chromatographic partial purification of MTs from cell homogenates with subsequent immuno-dot-blot assay, we showed that zinc pretreatment yielded a pronounced induction of MTs in HL-60 cells. We report that zinc-induced MTs were able to (i) completely bind intracellular copper, (ii) completely quench redox-cycling activity of copper, (iii) significantly inhibit copper-dependent oxidative stress in membrane phospholipids, and (iv) prevent copper-dependent apoptosis and its characteristic biochemical features (cytochrome c release from mitochondria into cytosol, caspase-3 activation, and externalization of phosphatidylserine in plasma membranes). In separate experiments, we used lung fibroblasts derived from MT1, MT2 knockout mice (MT(-)(/)(-)) and MT wild-type (MT(+/+)) mice. ZnCl(2) pretreatment resulted in a more than 10-fold induction of MTs in MT(+/+) cells, whereas the MT content in MT(-)(/)(-) cells remained low, at levels approximately 100-fold lower than in their MT wild-type counterparts. MT(-)(/)(-) cells were very sensitive to Cu-NTA and, most importantly, showed no response to ZnCl(2) pretreatment. In contrast, MT(+/+) cells were relatively more resistant to Cu-NTA, and this resistance was remarkably enhanced by ZnCl(2) pretreatment. Combined, our results demonstrate that metallothioneins function as effective antioxidants and an antiapoptotic mechanism in copper-challenged HL-60 cells.