We previously reported the isolation of T.DnaK.DnaJ chaperone complex from Thermus thermophilus. Here, we show that a novel factor is necessary for the assembly of T.DnaK and T.DnaJ into the complex. A dnaK gene cluster of T. thermophilus contained five genes, dnaK-grpE-dnaJ-orf4-clpB. Interestingly, T.DnaJ lacks the whole "cysteine-rich region" that has been postulated to be necessary to bind unfolded proteins. The orf4 gene encodes a novel 78-amino acid protein. Curiously, T.DnaK and T.DnaJ expressed in Escherichia coli did not form the complex. Careful reexamination of the T.DnaK.DnaJ complex revealed the presence of a small protein in the complex, which turned out to be a product of orf4. As expected, expression of three genes, dnaK-dnaJ-orf4, resulted in production of a T.DnaK.DnaJ complex in E. coli that was indistinguishable from the authentic complex in its ability to interact with nucleotide and denatured protein. The product of orf4 was also required for in vitro reconstitution of the complex and named T.DafA (T.DnaK.DnaJ assembly factor A). The complex comprises three copies each of T.DnaK, T.DnaJ, and T.DafA. Even though a definite homolog of T.DafA has not been found in the data base, this finding raises a possibility that interaction between DnaK and DnaJ chaperones in other organisms is also mediated by a small protein yet unnoticed.