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