This study elucidates the mechanism through which heat shock treatment influences the outcome of sepsis. Post-heat shock sepsis was induced in rats by CLP 24 h after whole-body hyperthermia. Liver cytosolic and nuclear fractions were collected and analyzed in early and late sepsis rats (sacrificed 9 and 18 h after CLP, respectively). During sepsis, levels of I-κB and nuclear factor-κB (NF-κB) declined in the cytosol of liver, whereas NF-κB increased in nucleus. NF-κB activity was significantly enhanced during sepsis, and the products of NF-κB target genes, such as TNF-α and inducible nitric oxide synthase (iNOS), were overexpressed. Heat shock treatment, inducing heat shock protein synthesis, prevented down-regulation of cytosolic I-κB and decreased translocation of NF-κB into the nucleus. Therefore, the sepsis-induced acceleration of NF-κB activation was inhibited. Expression of TNF-α and iNOS mRNA was also down-regulated. Coimmunoprecipitation with anti-NF-κB (p65) and anti-IκB antibodies verified an assembling phenomenon of heat shock protein (HSP) 72 with NF-κB and I-κB. We suggest that the mechanism preventing septic activation of NF-κB is that oversynthesized HSP72 forms a complex with NF-κB/I-κB, thus inhibiting nuclear translocation of NF-κB. HSP72 appears to play a crucial protective role in modulating the gene expression controlled by NF-κB in sepsis.