A major mammalian heat shock protein of 110 kDa (hsp110) has long been observed, but has not been cloned. We have cloned the hamster cDNA for hsp110 and show that it hybridizes on a Northern blot to a 3.5-kilobase heat-inducible message in hamster and mouse. The hsp110 sequence was found to share an approximately 30-33% amino acid identity with members of the hsp70 family, most of which occurs in the conserved ATP-binding domain of these molecules. In addition, five sequences were found to be highly similar to hsp110. These are the sea urchin egg receptor for sperm (Foltz, K.R., Partin, J. S., and Lennarz, W.J. (1993) Science 259, 1421-1425) and additional sequences from human and Caenorhaditis elegans and two from yeast. The carboxyl-terminal two-thirds of hsp110 and these five related proteins contain a pattern of highly conserved regions of sequence unique to this group. A probe containing these conserved sequences was found to strongly cross-react on a Southern blot with genomic sequences from yeast to man. A Western blot analysis of several murine tissues indicates that hsp110 is constitutively expressed in all mouse tissues and is highly expressed in brain. Therefore, hsp110 belongs to a new category of large and structurally unique stress proteins that are the most distantly related known members of the hsp70 family.