Previous studies have shown that a variety of mammalian cell types, including macrophages, contain small amounts of redox-active iron in their lysosomes. Increases in the level of this iron pool predispose the cell to oxidative stress. Limiting the availability of intralysosomal redox-active iron could therefore represent potential cytoprotection for cells under oxidative stress. In the present study we have shown that an initial 6 h exposure of J774 macrophages to 30 microM iron, added to the culture medium as FeCl3, increased the lysosomal iron content and their sensitivity to H2O2-induced (0.25 mM for 30 min) oxidative stress. Over time (24-72 h), however, the cells were desensitized to the cytotoxic effects of H2O2; most likely as a consequence of both lysosomal iron exocytosis and of ferritin synthesis (demonstrated by atomic absorption spectrophotometry, autometallography, and immunohistochemistry). When the cells were exposed to a second dose of iron, their lysosomal content of iron increased again but the cells became no further sensitized to the cytotoxic effects of H2O2. Using the lysosomotropic weak base, acridine orange, we demonstrated that after the second exposure to iron and H2O2, lysosomes remained intact and were no different from control cells which were exposed to H2O2 but not iron. These data suggest that the initial induction of ferritin synthesis leads to enrichment of lysosomes with ferritin via autophagocytosis. This limits the redox-availability of intralysosomal iron and, in turn, decreases the cells' sensitivity to oxidative stress. These in vitro observations could also explain why cells under pathological conditions, such as haemochromatosis, are apparently able to withstand high iron concentrations for some time in vivo.