Acute and chronic lung injury secondary to hyperoxia remains an important complication in critically ill patients, and, consequently, there is interest in developing strategies to protect the lung against hyperoxia. Heat shock proteins (HSPs) confer protection against a broad array of cytotoxic agents. In this study, we tested the hypothesis that increased expression of the 70-kDa HSP (HSP70) would protect cultured human respiratory epithelium against hyperoxia. Recombinant A549 cells were generated in which human HSP70 was increased by stable transfection with a plasmid containing human HSP70 cDNA under control of the cytomegalovirus promoter (A549-HSP70 cells). A549-HSP70 cells exposed to hyperoxia had greater acute survival rates and clonogenic capacity compared with wild-type A549 cells and with control cells stably transfected with the empty expression plasmid. Hyperoxia-mediated lipid peroxidation and ATP depletion were also attenuated in A549-HSP70 cells exposed to hyperoxia. Increased expression of HSP70 did not detectably alter mRNA levels of the intracellular antioxidants manganese superoxide dismutase, catalase, and glutathione peroxidase. Collectively, these data demonstrate a specific in vitro protective role for HSP70 against hyperoxia and suggest that potential mechanisms of protection involve attenuation of hyperoxia-mediated lipid peroxidation and ATP depletion.