The properties of molecular chaperones in protein-assisted refolding were examined in vitro using recombinant human cytosolic chaperones hsp90, hsc70, hsp70 and hdj-1, and unfolded beta-galactosidase as the substrate. In the presence of hsp70 (hsc70), hdj-1 and either ATP or ADP, denatured beta-galactosidase refolds and forms enzymatically active tetramers. Interactions between hsp90 and non-native beta-galactosidase neither lead to refolding nor stimulate hsp70- and hdj-1-dependent refolding. However, hsp90 in the absence of nucleotide can maintain the non-native substrate in a 'folding-competent' state which, upon addition of hsp70, hdj-1 and nucleotide, leads to refolding. The refolding activity of hsp70 and hdj-1 is effective across a broad range of temperatures from 22 degrees C to 41 degrees C, yet at extremely low (4 degrees C) or high (>41 degrees C) temperatures refolding activity is reversibly inhibited. These results reveal two distinct features of chaperone activity in which a non-native substrate can be either maintained in a stable folding-competent state or refolded directly to the native state; first, that the refolding activity itself is temperature sensitive and second, that hsp90, hsp70 (hsc70) and hdj-1 each have distinct roles in these processes.