Hydrogen peroxide-induced oxidative damage to nuclear DNA is supposed to involve Fenton-type chemistry with redox-active iron producing highly reactive hydroxyl radicals. Hydroxyl radicals are very short-lived and therefore act mainly at the site of their origin. To induce DNA damage by hydroxyl radical attack redox-active iron would have to be located close to DNA. The presence of free iron in the nucleus has never been convincingly shown. Recently, lysosomes as sites of intracellular digestion (e.g. of metallo proteins) were suggested as a pool of redox-active iron. The high molecular weight form (~ 80 kDa) of the iron chelator desferrioxamine (HMW-DFO) can only be taken up into cells by fluid-phase endocytosis resulting in an exclusively endosomal/lysosomal location of the HMW-DFO. Here we show that hydrogen peroxide induces lysosomal rupture and DNA damage in parallel and, more importantly, that both effects can be prevented by chelating lysosomal redox-active iron with HMW-DFO. We conclude that the lysosomal membrane is susceptible to rupture under oxidative stress leading to an efflux of redox-active iron into the cytoplasm. Part of this iron may relocate to the nucleus and induce DNA damage under continued oxidative stress.