The hexameric AAA+ (ATPase associated with various cellular activities) chaperone ClpB reactivates protein aggregates in collaboration with the DnaK system. An intriguing aspect of ClpB function is that the active hexamer is unstable and therefore questions how this chaperone uses multiple rounds of ATP hydrolysis to translocate substrates through its central channel. In the present paper, we report the use of biochemical and fluorescence tools to explore ClpB dynamics under different experimental conditions. The analysis of the chaperone activity and the kinetics of subunit exchange between protein hexamers labelled at different protein domains indicates, in contrast with the current view, that (i) ATP favours assembly and ADP dissociation of the hexameric assembly, (ii) subunit exchange kinetics is at least one order of magnitude slower than the ATP hydrolysis rate, (iii) ClpB dynamics and activity are related processes, and (iv) DnaK and substrate proteins regulate the ATPase activity and dynamics of ClpB. These data suggest that ClpB hexamers remain associated during several ATP hydrolysis events required to partially or completely translocate substrates through the protein central channel, and that ClpB dynamics is tuned by DnaK and substrate proteins.