Environmental changes, air pollution and ozone depletion are increasing oxidative stress, and global warming threatens health by heat stress. We now face a high risk of simultaneous exposure to heat and oxidative stress. However, there have been few studies investigating their combined adverse effects on cell viability. Pretreatment of hydrogen peroxide (H(2)O(2)) specifically and highly sensitized cells to heat stress, and enhanced loss of mitochondrial membrane potential. H(2)O(2) exposure impaired the HSP40/HSP70 induction as heat shock response (HSR) and the unfolded protein recovery, and enhanced eIF2alpha phosphorylation and/or XBP1 splicing, land marks of ER stress. These H(2)O(2)-mediated effects mimicked enhanced heat sensitivity in HSF1 knockdown or knockout cells. Importantly, thermal preconditioning blocked H(2)O(2)-mediated inhibitory effects on refolding activity and rescued HSF1 +/+ MEFs, but neither blocked the effects nor rescued HSF1 -/- MEFs. These data strongly suggest that inhibition of HSR and refolding activity is crucial for H(2)O(2)-mediated enhanced heat sensitivity. H(2)O(2) blocks HSR and refolding activity under heat stress, thereby leading to insufficient quality control and enhancing ER stress. These uncontrolled stress responses may enhance cell death. Our data thus highlight oxidative stress as a crucial factor affecting heat tolerance.