Thermus thermophilus holo-chaperonin (holo-cpn), a cpn60 14-mer-cpn10 7-mer complex, which was overproduced in Escherichia coli, was easily purified from a crude cell extract by heat treatment. The refolding of guanidine hydrochloride (GdnHCl) unfolded enzymes in the absence and presence of T. thermophilus holo-cpn was studied using Leuconostoc mesenteroides glucose-6-phoshate dehydrogenase (G6PD) and bovine deoxyribonuclease I (DNase I) as model enzymes. T. thermophilus holo-cpn significantly enhanced the recovery of the enzyme activities at around 30-40 degrees C, which is the same temperature range within which the enzymes spontaneously recovered their highest activity. Since the complex formed by T. thermophilus cpn60 and cpn10 was stable, it could be separated from G6PD and DNase I in the refolding mixture by ultrafiltration using membranes with molecular weight cut-offs of 300 kDa and 100 kDa, respectively, and successfully reused. To increase the final concentration of renatured enzyme, step-wise addition of unfolded enzyme into the refolding mixture containing T. thermophilus holo-cpn was found to be effective. A combination of step-wise addition of unfolded enzyme and recycling of T. thermophilus holo-cpn using the ultrafiltration system significantly increased both the recovery of enzyme activity and the final concentration of renatured enzyme.