Oxidative injury to cells such as the retinal pigment epithelium (RPE) is often modeled using H(2)O(2)-treated cultures, but H(2)O(2) concentrations are not sustained in culture medium. Here medium levels of H(2)O(2) and cytotoxicity were analyzed in ARPE-19 cultures after H(2)O(2) delivery as a single pulse or with continuous generation using glucose oxidase (GOx). When added as a pulse, H(2)O(2) is rapidly depleted (within 2 h); cytotoxicity at 24 h, determined by the MTT assay for mitochondrial function, is unaffected by medium replacement at 2 h. Continuous generation of H(2)O(2) produces complex outcomes. At low GOx concentrations, H(2)O(2) levels are sustained by conditions under which generation matches depletion, but when GOx concentrations produce cytotoxic levels of H(2)O(2), oxidant depletion accelerates. Acceleration results partly from the release of contents from oxidant-damaged cells as indicated by testing depletion after controlled membrane disruption with detergents. Cytotoxicity analyses show that cells can tolerate short exposure to high H(2)O(2) doses delivered as a pulse but are susceptible to lower chronic doses. The results provide broadly applicable guidance for using GOx to produce sustained H(2)O(2) levels in cultured cells. This approach will be specifically useful for modeling chronic stress relevant to RPE aging and have a wider value for studying cellular effects of sublethal oxidant injury and for evaluating antioxidants that may protect significantly against mild but not lethal stress.