Coupling with ATP hydrolysis and cooperating with GroES, the double ring chaperonin GroEL assists the folding of other proteins. Here we report novel GroEL-GroES complexes formed in fluoroberyllate (BeFx) that can mimic the phosphate part of the enzyme-bound nucleotides. In ATP, BeFxstops the functional turnover of GroEL by preventing GroES release and produces a symmetric 1:2 GroEL-GroES complex in which both GroEL rings contain ADP·BeFxand an encapsulated substrate protein. In ADP, the substrate protein-loaded GroEL cannot bind GroES. In ADPplusBeFx, however, it can bind GroES to form a stable 1:1 GroEL-GroES complex in which one of GroEL rings contains ADP·BeFxand an encapsulated substrate protein. This 1:1 GroEL-GroES complex is converted into the symmetric 1:2 GroEL-GroES complex when GroES is supplied in ATPplusBeFx. Thus, BeFxstabilizes two GroEL-GroES complexes; one with a single folding chamber and the other with double folding chambers. These results shed light on the intermediate ADP·Pinucleotide states in the functional cycle of GroEL.