CHAPERONINS are king-shaped protein complexes that are essential in the cell, mediating ATP-dependent polypeptide folding in a variety 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 properties of GroEL to its crystal structure(7). Our functional tests identify a putative polypeptide-binding site on the inside surface of the apical domain, facing the central channel, consisting of hydrophobic 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.