Our laboratory has been characterizing the hsp70 multigene family from the nematode Caenorhabditis elegans as the first step to the genetic characterization of the heat shock response in a relatively simple multicellular eukaryote. Two gene members, hsp-1 and hsp-2ps have already been characterized (Snutch et al., 1988; Heschl and Baillie, 1989). The third gene member, hsp-3, is expressed constitutively and is non-heat inducible; its mRNA is most abundant at the L1 larval stage. The hsp-3 protein (hsp70C) shares a high degree of identity with the rat grp78 protein and has a long, hydrophobic leader sequence. The carboxyl terminus of hsp70C has the putative ER-retention signal, KDEL. The fourth gene member, hsp-6 is expressed constitutively and moderately heat inducible. A partial hsp-6 protein (hsp70F) sequence shares a higher degree of identity with the Escherichia coli dnaK protein than with eukaryotic hsp70 proteins. The predicted amino-terminal half of the hsp70F polypeptide also contains a long, amphiphilic leader sequence similar to mitochondrial import leader sequences. These two genes encode proteins that potentially cross intracellular membranes. We compared the 5'-flanking DNA from the C. elegans hsp-3 gene to fragment containing enhancer activity from the rat grp78 gene regulatory region (Lin et al., 1986). A 23-nucleotide sequence was conserved between the two promoter regions. This sequence shares approximately 80% identity between these two evolutionary distant organisms. A comparison to other hsp70 genes did not reveal any conservation of this 23-nucleotide sequence. We propose that this sequence may be involved in a unique aspect of the regulation of the C. elegans' grp78-like gene and the rat grp78 gene.