Preischemic treatment with atrial natriuretic peptide (ANP) attenuates ischemia-reperfusion injury of the rat liver via cyclic guanosine monophosphate (cGMP). The attenuated activation of nuclear factor κB (NF-κB) seems to contribute to this effect. The aim of this study was to determine whether heat shock proteins are involved in these molecular pathways. Livers of male Sprague-Dawley rats were continuously perfused with Krebs-Henseleit (KH) buffer with or without ANP or 8-Br-cGMP. In different experiments livers were perfused with or without ANP for 20 minutes, kept in cold storage solution for 24 hours, and reperfused. Activation of heat shock transcription factor (HSF) (by electrophoretic mobility shift assay), heat shock protein 70 (HSP70), and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA (by reverse transcription polymerase chain reaction [RT-PCR]), as well as HSP70 (by Western blot) were investigated in freeze-clamped liver samples. During continuous perfusion ANP as well as 8-Br-cGMP activated HSF, HSP70 protein concentrations paralleled HSF-activation. ANP pretreated livers exhibited elevated HSF after 24 hours of ischemia and elevated HSP70 mRNA levels during reperfusion. ANP prevented the marked decrease of HSP70 protein during reperfusion. Coimmunoprecipitation studies showed increased binding of HSP70 to inhibitory factor κB (IκB) in ANP-treated livers. In conclusion, we showed the cGMP-mediated activation of HSF by ANP, which resulted in elevated HSP70 mRNA and protein concentrations and correlated with enhanced binding of HSP70 to IκB. This could be an important mechanism of ANP-mediated prevention of hepatic preservation damage.