A polymerase chain reaction (PCR) was developed for identification of Borrelia burgdorferi in biological specimens. The diagnostic efficiency was compared with that of in vitro culture. A primer set specifying a 791-bp DNA fragment of the B. burgdorferi B31 flagellin gene was used. Amplified DNA sequences were analyzed by agarose gel electrophoresis, and the identity of amplified DNA was confirmed by restriction enzyme cleavage and Southern blot hybridization with a 32P-labeled probe. By using purified B. burgdorferi DNA, the detection limit of the assay was approximately 0.002 pg of DNA, corresponding to one copy of the B. burgdorferi genome. By using in vitro-cultivated B. burgdorferi without prior DNA purification as the template DNA, 2 to 20 organisms could be detected. A 791-bp DNA fragment was amplified from all of 18 different B. burgdorferi strains tested, as well as from Borrelia hermsii and Borrelia anserina but not from Treponema pallidum. The efficacy of the PCR assay was evaluated on spleen, renal, and urinary bladder tissue specimens from eight experimentally infected gerbils. Specimens from the same organs were cultured in BSK medium in parallel. Of 24 organs, 21 (88%) were PCR positive and 17 (71%) were culture positive. All culture-positive specimens were also PCR positive. Compared with B. burgdorferi cultivation, PCR had at least a comparable diagnostic sensitivity, it was less laborious, and results were available within 1 to 2 days.