Amide hydrogen exchange (HX) in combination with mass spectrometry (MS) is a powerful tool to analyze the folding and dynamics of proteins. In the traditional methodology the exchange time is controlled by manual pipetting, thereby limiting the time resolution to several seconds. Some conformational changes in proteins, however, occur in the subsecond time scale, making it desirable to perform HX at shorter time intervals down to the limit set by the intrinsic chemical exchange rate. We now report the development of the first completely on-line quenched-flow setup that allows the performance of HX experiments in the 100-sec to 30-sec time scale, on-line proteolytic digestion using immobilized proteases, rapid desalting, and MS analysis. We show that conformational fluctuations in the range of seconds can be detected and protection factors as small as 10 reproducibly determined. Using this setup we investigated the conformational properties of Escherichia coli heat-shock transcription factor sigma32 free in solution. Our results indicate that the C-terminal sigma4 domain of sigma32, which is responsible for the recognition of the -35 region of heat shock promoters, contains more extensive secondary structure than expected when compared with the structure of the homologous sigma-factor sigmaA in complex with the RNA-polymerase. This setup should be very useful for a more accurate analysis of structural motions in proteins in the subsecond to second time scale relevant to allostery and enzyme function.