Stress conditions such as heat shock alter the transcriptional profile in all organisms. In Escherichia coli the heat shock transcription factor, sigma 32, out-competes upon temperature up-shift the housekeeping sigma-factor, sigma 70, for binding to core RNA polymerase and initiates heat shock gene transcription. To investigate possible heat-induced conformational changes in sigma 32 we performed amide hydrogen (H/D) exchange experiments under optimal growth and heat shock conditions combined with mass spectrometry. We found a rapid exchange of around 220 of the 294 amide hydrogens at 37 degrees C, indicating that sigma 32 adopts a highly flexible structure. At 42 degrees C we observed a slow correlated exchange of 30 additional amide hydrogens and localized it to a helix-loop-helix motif within domain sigma 2 that is responsible for the recognition of the -10 region in heat shock promoters. The correlated exchange is shown to constitute a reversible unfolding with a half-life of about 30 min due to a temperature-dependent decrease in stabilization energy. We propose that this gradual decrease in stabilization energy of domain sigma 2 with increasing temperatures facilitates the unfolding of sigma 32 by the AAA+ protease FtsH thereby decreasing its half-life. Taken together our data show that the sigma 2 domain of sigma 32 can act as a thermosensor, which might be important for the heat shock regulation.