AbstractThe proteasome has a crucial part in the degradation of normal, damaged, mutant or misfolded proteins within both the ubiquitin ATP‐dependent and the ubiquitin ATP‐independent pathways. Proteasome‐mediated proteolysis is modulated by diverse factors, and in this regard, chaperonins have been attracting great interest. The investigation on the role of a co‐chaperonin, namely GroES, in the modulation of proteasomal activity was the focus of this work. Our study reports on an analytical approach based on combined fluorimetric, chromatographic (applied to the enzymatic activity evaluation), surface plasmon resonance techniques and molecular modelling, addressed to the assessment and characterization of the interaction. Globally, we described a high affinity interaction between GroES and two different 20 S (immuno‐ and constitutive) proteasomes, uncovering new scenarios on their possible physio‐pathological role, specifically on the ability of proteasomes to interact both with unfolding and folding‐ assisting macromolecules. Copyright © 2008 John Wiley & Sons, Ltd.