The eukaryotic cytosolic chaperonin, CCT, plays an essential role in mediating ATP-dependent folding of actin and tubulin. There is debate about whether it mediates folding through a single round of association followed by release of native forms, or through cycles of binding and full release in which only a fraction of released molecules reaches native form in any cycle. We examine the fate of newly synthesized substrate proteins bound to CCT in reticulocyte lysate or intact Xenopus oocytes. When a chaperonin "trap," able to bind but not release substrate protein, is introduced, production of the native state is strongly inhibited, associated with transfer to trap. While predominantly nonnative forms of actin, tubulin, and a newly identified substrate, G(alpha)-transducin, are released from CCT, a small fraction reaches native form with each round of release, inaccessible to trap. This overall mechanism resembles that of the bacterial chaperonin, GroEL.