Radiation stimulates signaling cascades that result in the activation of several transcription factors that are believed to play a central role in protective response(s) to ionizing radiation (IR). It is also well established that heat shock alters the regulation of signaling cascades and transcription factors and is a potent radiosensitizing agent. To explore the hypothesis that heat disrupts or alters the regulation of signaling factors activated by IR, the effect of heat shock on IR-induced activation of NF-κB was determined. Irradiated HeLa cells demonstrated transient increases in NF- κB DNA binding activity and NF-κB protein nuclear localization. In addition, irradiated cells demonstrated increased I-κB phosphorylation and decreased IκBα cytoplasmic protein levels, corresponding temporally with the increase of NF-κB DNA binding. Heat shock prior to IR inhibited the increase in NF-κB DNA binding activity, nuclear localization of NF-κB, and the phosphorylation and subsequent degradation of I-κB. I-κB kinase (IKK) immunoprecipitation assays demonstrated an increase in IKK catalytic activity in response to IR that was inhibited by pretreatment with heat. Kinetic experiments determined that heat-induced inhibition of NF-κB activation in response to IR decayed within 5 h after heating. Furthermore, pretreatment with cycloheximide, to block de novo protein synthesis, did not alter heat shock inhibition of IR induction of NF-κB. These experiments demonstrate that heat shock transiently inhibits IR induction of NF-κB DNA binding activity by preventing IKK activation and suggests a mechanism independent of protein synthesis.