The chaperonins GroEL and GroES of Escherichia coli facilitate protein folding in an adenosine triphosphate (ATP)-dependent reaction cycle, The kinetic parameters for the formation and dissociation of GroEL-GroES complexes were analyzed by surface plasmon resonance. Association of GroES and subsequent ATP hydrolysis in the interacting GroEL toroid resulted in the formation of a stable GroEL:ADP:GroES complex. The complex dissociated as a result of ATP hydrolysis in the opposite GroEL toroid, without formation of a symmetrical GroEL:(GroES)(2) intermediate. Dissociation was accelerated by the addition of unfolded polypeptide. Thus, the functional chaperonin unit is an asymmetrical GroEL:GroES complex, and substrate protein plays an active role in modulating the chaperonin reaction cycle.