Enhanced survival of both individual cells and whole organisms following a heat stress is termed thermotolerance. In organisms, the maintenance of tissue function rather than the survival of individual cells ultimately determines outcome following thermal challenge. We used MDCK kidney epithelial cells to compare alterations in chaperone activity (as a measure of cellular tolerance) and epithelial barrier function (as a measure of physiological tolerance) after thermal challenge. Quercetin, an inhibitor of heat shock factor–dependent transcriptional activity, both potentiated the effects of heat on naive monolayers and blocked conditioning of monolayers following moderate heat shock, suggesting a central role of heat shock protein (HSP) family members in the maintenance of epithelial integrity. We used MDCK cells that constitutively overexpressed HSP70 to demonstrate 2 functionally distinct components of the response of monolayers to thermal stress. The maintenance of epithelial barrier function during exposure to elevated temperatures is regulated by a complex network of processes that involve the actions of HSP70 but that are independent of alterations in chaperone activity as reflected by changes in the thermal inactivation/refolding of luciferase. In contrast, the restoration of barrier function following a heat stress is directly modulated by HSP70 in a manner that can be fully accounted for by changes in chaperone activity. This study demonstrates an important, albeit complex, protective role for heat shock proteins in the modulation of MDCK epithelial barrier function following a thermal stress.