PROTEIN folding mediated by the molecular chaperone GroEL occurs by its binding to non-native polypeptide substrates and is driven by ATP hydrolysis(1). Both of these processes are influenced by the reversible association of the co-protein, GroES (refs 2-4). GroEL and other chaperonin 60 molecules(5) are large, cylindrical oligomers consisting of two stacked heptameric rings of subunits(6,7); each ring forms a cage-like structure(8) thought to bind polypeptides in a central cavity(8-10). Chaperonins play a passive role in folding by binding or sequestering folding proteins to prevent their aggregation(11-13), but they may also actively unfold substrate proteins trapped in misfolded forms, enabling them to assume productive folding conformations(14-16). Biochemical studies show that GroES improves the efficiency of GroEL function(2,3,17), but the structural basis for this is unknown. Here we report the first direct visualization, by cryo-electron microscopy, of a non-native protein substrate (malate dehydrogenase) bound to the mobile, outer domains at one end of GroEL. Addition of GroES to GroEL in the presence of ATP causes a dramatic hinge opening of about 60 degrees. GroES binds to the equivalent surface of the GroEL outer domains, but on the opposite end of the GroEL oligomer to the protein substrate.