The chaperonin proteins, groEL and groES belong to a ubiquitous sub-family of heat-shock molecular chaperones, found in prokaryotes and in eukaryotic organelles. The chaperonins assist the folding of nascent, organelle-imported or stress-destabilized polypeptides. Using the photosynthetic enzyme Rubisco as a folding substrate, we demonstrate in vitro that this process requires firstly the formation of a binary-complex between an unstable Rubisco folding-intermediate with the oligomeric form of groEL. The second step, resulting in the formation of active Rubisco, consists of an ATP- and K+-dependent discharge of the groEL-Rubisco binary complex. GroES is required as a coupling factor between the ATP hydrolysis and the Rubisco refolding reactions which take place on the groEL oligomer. At 25-degrees-C, chaperonins assist Rubisco refolding by preventing its aggregation and not by rescuing misfolded proteins.