A highly specific and sensitive assay for Borrelia burgdorferi, the causative agent of Lyme disease, was developed using the polymerase chain reaction (PCR). The target DNA sequence was of chromosomal origin and conserved, by hybridization analyses, among all strains of B. burgdorferi tested but was not present in the most closely related member of the genus, B. hermsii. The PCR assay developed from this sequence reacted with 17 of 18 strains of B. burgdorferi but not with any other Borrelia species tested. The assay was sensitive to fewer than five copies of the B. burgdorferi genome, even in the presence of a 10(6)-fold excess of eukaryotic DNA. This assay should greatly facilitate the accurate diagnosis of Lyme disease and provide a means with which to investigate the pathogenesis, transmission, and basic biology of B. burgdorferi.