CHAPERONINS are ring-shaped protein complexes that are essential in the cell, mediating ATP-dependent polypeptide folding in a vari-ety of compartments^1–3. Recent studies suggest that they function through multiple rounds of binding and release of non-native proteins: with each round of ATP-driven release into the bulk solution, a substrate protein kinetically partitions between folding to the native state or rebinding to another chaperonin molecule^4–6. To gain further insight into the mechanism of polypeptide binding and release by the chaperonin GroEL from Escherichia coli, we have undertaken a mutational analysis that relates the functional prop-erties of GroEL to its crystal structure⁷. Our functional tests iden-tify a putative polypeptide-binding site on the inside surface of the apical domain, facing the central channel, consisting of hydropho-bic residues. These same residues are essential for binding of the co-chaperonin GroES, which is required for productive polypeptide release. A highly conserved residue, Asp 87, positioned within a putative nucleotide-binding pocket in the top of the equatorial domain, is essential for ATP hydrolysis and polypeptide release.