The 170 kDa glucose-regulated protein (grp170) is an endoplasmic reticulum resident protein that shares some sequence homology with both the hsp70 and hsp110 heat shock protein (hsp) families, yet is representative of a third and unique family of stress proteins. Despite observations indicating important roles in normal cellular functions, the in vitro chaperone properties of grp170 have not been rigorously examined. We have cloned mouse grp170 and expressed the recombinant protein in a baculovirus expression system. The function of recombinant grp170 was then assessed by determining its ability to bind to and prevent aggregation of heat-denatured luciferase. Grp170 maintains heat-denatured luciferase in a soluble state in the absence of ATP. In the presence of rabbit reticulocyte lysate, grp170 can refold and partially restore function to denatured luciferase. The chaperoning function of grp170 was also studied using domain deletion mutants, designed using the crystal structure of DnaK and the theoretical secondary structure of hsp110 as guides. Unlike hsp70 and hsp110, grp170 appears to have two domains capable of binding denatured luciferase and inhibiting its heat-induced aggregation. The two domains were identified as being similar to the classical beta-sandwich peptide binding domain and the C-terminal alpha-helical domain in hsp70 and hsp110. The ability of the C-terminal region to bind peptides is a unique feature of grp170.