Because efficient folding in vivo and reconstitution in vitro of phage P22 tailspike protein is temperature-sensitive, and because a chaperone function of the GroE proteins for tailspike folding in vivo has been suggested by genetic observations, the interactions of purified Escherichia coli GroE proteins with phage P22 tailspikes during refolding in vitro were investigated. At elevated temperature (> 30 degrees C), in the absence of ATP, GroEL effectively trapped refolding tailspike protein and prevented reconstitution. Tailspike protein was released from GroEL by addition of ATP around 35 degrees C or without added ATP upon cooling to 25 degrees C, and native tailspike trimers were formed. In accordance with the cold release, tailspike reconstitution at < or = 25 degrees C was unaffected by GroE. No formation of native tailspike trimers was observed, when refolding was initiated at 42 degrees C in the presence of the GroE proteins and ATP or when tailspike protein was dissociated from a preformed complex with the chaperone by addition of ATP at 42 degrees C. In contrast to other GroE ligands, the tailspike polypeptide was bound by and released from GroE in similar states of folding, and the presence of GroES in addition to GroEL had no effect on reconstitution yields at any temperature. Thus, the GroE proteins may exhibit widely differing interactions even with proteins showing similarly temperature-sensitive yields of folding.