The 110 kDa heat shock protein (HSP) (hsp110) has been shown to be a diverged subgroup of the hsp70 family and is one of the major HSPs in mammalian cells [1,2]. In examining the native interactions of hsp110, we observed that it is found to reside in a large molecular complex. Immunoblot analysis and co-immunoprecipitation studies identified two other HSPs as components of this complex, hsc70 and hsp25. When examined in vitro, purified hsp25, hsp70 and hsp110 were observed to spontaneously form a large complex and to directly interact with one another. When luciferase was added to this in vitro system, it was observed to migrate into this chaperone complex following heat shock. Examination of two deletion mutants of hsp110 demonstrated that its peptide-binding domain is required for interaction with hsp25, but not with hsc70. The potential function of the hsp110-hsc70-hsp25 complex is discussed.