Most protein mutations, and mutations that alter protein functions in particular, undermine stability and are therefore deleterious. Chaperones, or heat-shock proteins, are often implicated in buffering mutations, and could thus facilitate the acquisition of neutral genetic diversity and the rate of adaptation. We examined the ability of theEscherichia coliGroEL/GroES chaperonins to buffer destabilizing and adaptive mutations. Here we show that mutational drifts performedin vitrowith four different enzymes indicated that GroEL/GroES overexpression doubled the number of accumulating mutations, and promoted the folding of enzyme variants carrying mutations in the protein core and/or mutations with higher destabilizing effects (destabilization energies of >3.5 kcal mol-1, on average, versus ∼1 kcal mol-1 in the absence of GroEL/GroES). The divergence of modified enzymatic specificity occurred much faster under GroEL/GroES overexpression, in terms of the number of adapted variants (≥2-fold) and their improved specificity and activity (≥10-fold). These results indicate that protein stability is a major constraint in protein evolution, and buffering mechanisms such as chaperonins are key in alleviating this constraint.