Rat heart-derived H9c2 myoblasts were preconditioned by heat or metabolic stress followed by recovery under normal conditions. Then cells were subjected to severe ATP depletion and the stress-associated proteotoxicity was assessed on (i) the increase in a Triton X-100-insoluble component of total cellular protein and (ii) the rate of inactivation and insolubilization of transfected luciferase with cytoplasmic or nuclear localization. Both heat and metabolic preconditioning elevated the intracellular HSP70 level and reduced cell death after sustained ATP depletion without affecting the rate and extent of ATP decrease. Each preconditioning attenuated the stress-induced insolubility among total cellular protein as well as the inactivation and insolubilization of cytoplasmic and nuclear luciferase. Transient overexpression of human HSP70 in cells also attenuated both the cytotoxic and proteotoxic effects of ATP depletion. Quercetin, a blocker of stress-responsive HSP expression, abolished the effects of stressful preconditioning, while it did not influence the effects of the HSP70 overexpression. Analyses of the cellular fractions revealed that both the stress-preconditioned and the HSP70-overexpressing cells longer retain the soluble pool of HSP70 during ATP depletion. Herein, larger amounts of other proteins co-immunoprecipitated with excess HSP70 from the soluble fractions, as compared with control cells deprived of ATP. It is concluded that excess HSP70 can protect ATP-depleted cells against protein aggregation in both cytoplasmic and nuclear compartments. Moreover, this is the first demonstration of positive correlation between chaperone activity within cells and their viability in the context of ischemia-like stress.