Heat shock (HS) treatment has been previously shown to suppress the IkappaB/nuclear factor-kappaB (NF-kappaB) cascade by denaturing, and thus inactivating IkappaB kinase (IKK). HS is characterized by the induction of a group of heat shock proteins (HSPs). However, their role in the HS-induced suppression of the IkappaB/NF-kappaB cascade is unclear. Adenovirus-mediated HSP70 overexpression was found not to suppress the TNF-alpha-induced activation of the IkappaB/NF-kappaB pathway, thus suggesting that HSP70 is unlikely to suppress this pathway. When TNF-alpha-induced activation of the IkappaB/NF-kappaB pathway was regained 24 h after HS, HSP70 was found to be highly up-regulated. Moreover, blocking HSP70 induction delayed TNF-alpha-induced IkappaBalpha degradation and the resolubilization of IKK. In addition, HSP70 associated physically with IKK, suggesting that HSP70 is involved in the recovery process via molecular chaperone effect. Adenovirus-mediated HSP70 overexpression prior to HS blocked the IkappaBalpha stabilizing effect of HS by suppressing IKK insolubilization. Moreover, the up-regulation of endogenous HSP70 by preheating, suppressed this subsequent HS-induced IKK insolubilization, and this effect was abrogated by blocking HSP70 induction. These findings indicate that HSP70 accumulates during HS and negatively regulates the HS-induced suppression of the IkappaB/NF-kappaB cascade by facilitating the renaturation of IKK and blocking its further denaturation.