Borrelia burgdorferi sensu stricto homologues of cheA and cheW were cloned and characterized. A combination of strategies such as polymerase chain reaction (PCR) using degenerate primers, random-primed gene walking PCR and construction of a h library were used to identify the putative cheA gene. Sequence analysis of the DNA fragments obtained from the CT strain identified a 2,592-bp open reading frame (ORF) encoding an 864-amino-acid protein with significant similarity (53-64.6% identical residues) to the CheA of several genera of eubacteria. In particular, hallmarks of a histidine kinase family were found such as the location of the histidine autophosphorylation domain very close to the NH2 terminus and the nucleotide-binding site. A second ORF located immediately downstream from the putative borrelial cheA gene encoded a 195-amino-acid protein which displayed a high level of similarity to bacterial CheW. Using reverse transcription PCR, we demonstrated that cheA and cheW form an operon with an upstream, unidentified ORF. The cheA and cheW homologues were located at 722-737 kbp, 738-768 kbp and 743-824 kbp on the linear chromosomes of B. burgdorferi sensu stricto, B. garinii and B. afzelii respectively. Identification of cheA and cheW is the first step toward elucidation of a possible role of chemotaxis in virulence of the Lyme disease borreliae.