To test for redox regulation of anion channels in erythroid cells, we exposed K562 cells to oxidants and measured changes in transmembrane Cl(-) currents using patch-clamp, and in intracellular Cl(-) content using the Cl(-) selective dye MQAE. Oxidation with tert-butylhydroperoxide or H(2)O(2) produced a plasma membrane anion permeability with a permselectivity of NO(3)(-)>lactate(-)>gluconate(-). The permeability increase was paralleled by insertion of ClC-3 protein into the plasma membrane as evident from immunofluorescence microscopy and surface biotinylation. Down-regulation of ClC-3 protein by RNA interference as assessed by immunoblotting decreased the oxidation-stimulated permeability. In conclusion, oxidation induces surface expression of ClC-3 and activation of a ClC-3-dependent anion permeability in K562 cells.