For studying the possible interaction between the chaperonins and phosphofructokinase (PFK), bacterial chaperonins GroEL, GroES, and the PFK were co-purified from chaperonin over-expressing, heat treated E. coli strains. GroEL interacted with PFK in the presence of Mg2+, leading to a gradual decrease in the activity of the enzyme. This type of GroEL-PFK interaction was overcame by the GroES. On the effect of the addition of ATP to the GroEL-PFK complex, the decreased activity of the enzyme was recovered suggesting release of the GroEL-bound enzyme. Contrary to this, in a complete refolding system containing GroEL, GroES, ATP and Mg2+, activity of heat treated bacterial PFK gradually increased showing, that GroEL and GroES together play a role in the folding and/or assembly of PFK. Similar effect of refolding system was also observed on rabbit muscle PFK. The data show that PFK can interact with GroEL, which results in binding of the enzyme; by contrast, GroEL and GroES together has a folding effect leading ultimately to increase of the activity of the enzyme.