Heat shock protein-70 (Hsp70) is the main heat-inducible member of the 70-kDa family of chaperones that assist cells in maintaining proteins functional under stressful conditions. In the present investigation, the role of Hsp70 in the molecular mechanism of hydrogen peroxide-induced DNA damage to HeLa cells in culture was examined. Stably transfected HeLa cell lines, overexpressing or lacking Hsp70, were created by utilizing constitutive expression of plasmids containing the functional hsp70 gene or hsp70-siRNA, respectively. Compared to control cells, the Hsp70-overexpressing ones were significantly resistant to hydrogen peroxide-induced DNA damage, while Hsp70-depleted cells showed an enhanced sensitivity. In addition, the "intracellular calcein-chelatable iron pool" was determined in the presence or absence of Hsp70 and found to be related to the sensitivity of nuclear DNA to H2O2. It seems likely that the main action of Hsp70, at least in this system, is exerted at the lysosomal level, by protecting the membranes of these organelles against oxidative stress-induced destabilization. Apart from shedding additional light on the mechanistic details behind the action of Hsp70 during oxidative stress, our results indicate that modulation of cellular Hsp70 may represent a way to make cancer cells more sensitive to normal host defense mechanisms or chemotherapeutic drug treatment. © 2006 Elsevier Inc. All rights reserved.