Rhizobium fredii strain USDA257 does not nodulate soybean (Glycine max (L.) Merr.) cultivar McCall. Mutant 257DH5, which contains a Tn5 insert in the bacterial chromosome, forms nodules on this cultivar, but acetylene-reduction activity is absent. We have sequenced the region corresponding to the site of Tn5 insertion in this mutant and find that it lies within a 1176bp open reading frame that we designate nolC. nolC encodes a protein of deduced molecular weight 43564. Nucleotide sequences homologous to nolC are present in several other Rhizobium strains, as well as Agrobacterium tumefaciens, but not in Pseudomonas syringae pathovar glycinea. nolC lacks significant sequence homology with known genes that function in nodulation, but is 61% homologous to dnaJ, an Escherichia coli gene that encodes a 41 kDa heat-shock protein. Both R. fredii USDA257 and mutant 257DH5 produce heat-shock proteins of 78, 70, 22, and 16kDa. A 4.3kb EcoRI-HindIII subclone containing nolC expresses a single 43kDa polypeptide in mini-cells. A longer, 9.4kb EcoRI fragment expresses both the 43kDa polypeptide and a 78kDa polypeptide that corresponds in size to that of the largest heat-shock protein. Thus, although nolC has strong sequence homology to dnaJ and appears to be linked to another heat-shock gene, it does not directly function in the heat-shock response.