MjHsp16.5 was separately labeled by fluorescent dye Cy3 and Cy5.5. The dissociation event ofa single 24-mer MjHsp16.5 molecule was captured by single-molecule imaging (SMI). Temperature-regulatedsubunit exchange was revealed by the real-time fluorescence resonance energy transfer (FRET). Thecombination of single-molecular statistics and kinetic parameters from FRET experiments leads to theconclusion that below 75 °C the rate-determining step of the subunit exchange was the dissociation of thedye-labeled 24-mer in which the dimer was intact, whereas above 75 °C, smaller units emerged in theexchange and the rate-determining step had the character of a bimolecular reaction.