The gene encoding an immunoreactive Borrelia burgdorferi HSP70 homolog was isolated and characterized. The predicted amino acid sequence of this spirochetal protein confirms that this gene encodes a member of the HSP70 family of proteins. Although there appears to be a single copy of this gene on the spirochetal chromosome, two distinct transcripts hybridizing to the hsp70 probe are detected in RNA isolated from B. burgdorferi. The amount of spirochetal HSP70 RNA transcripts is shown to be thermally regulated. Antibodies in the serum of three Lyme arthritis patients and cloned T-cell lines isolated from one patient with Lyme arthritis recognize the expressed recombinant HSP70, indicating that it is an immunologically important spirochetal antigen. Antibodies in a rabbit antiserum, as well as antibodies in the serum of two of three Lyme arthritis patients examined, bound to expressed truncated recombinant HSP70s with 250 amino acids deleted from either the amino or carboxy terminus of the protein. However, antibodies in the serum of three Lyme arthritis patients, which were reactive with spirochetal HSP70, did not cross-react with human HSP70 proteins.