The guanidine-hydrochloride (Gdn-HCl) and thermally induced unfolding of Hsp70 from Meiothermus ruber (Mru.Hsp70) were analyzed using tryptophan fluorescence and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. The ANS binding to Mru.Hsp70 showed both the increase in fluorescence intensity and a shift in emission maximum Anal. of the unfolding profile of Mru.Hsp70 indicated that Gdn-HCl induced unfolding of Mru.Hsp70 occurred through intermediate species. The tryptophan and ANS fluorescence emission spectra revealed that ATP induced conformational change increased the thermal stability of Mru.Hsp70. The data obtained are similar to those of Escherichia coli DnaK. The ATPase activity of chaperones is fundamental for their biol. activity. It this paper we demonstrate that, in contrast to Thermus thermophilus, both Mru.Hsp40 and Mru.Hsp22 co-chaperones affect the ATPase activity of Mru.Hsp70. The use of truncated Mru.Hsp40 proteins showed that full-length Mru.Hsp40 is required for stimulation of ATPase activity of Mru.Hsp70. E. coli GrpE could act as nucleotide exchange factor the in thermophilic Hsp70 ATP hydrolysis reaction. However, the role of E. coli DnaJ in the M. ruber ATP cycle needs further anal. We selected the new substrate laccase A (laccA) suitable for determination of refolding activity of thermophilic chaperones.