We investigated the use of the polymerase chain reaction (PCR) to detect Borrelia burgdorferi strain B-31 in human blood and urine experimentally inoculated with 5 and 1 borreliae/cm3, respectively, and to biotinylate a DNA probe specific for B. burgdorferi in the dot blot and Southern blot assays. When the blood and urine samples were subjected to PCR, a 370-bp amplified product was consistently visible on agarose gel electrophoresis after 30 and 45 cycles, respectively. The total human genomic DNA extracted from a 1-cm3 sample of inoculated blood was approximately 6.25 micrograms, and the total amount of B. burgdorferi DNA was estimated to be 0.01 pg/6.25 micrograms of the human DNA. For PCR, 2.5 micrograms of human DNA which contained the equivalent of 0.004 pg of borrelia DNA (approximately two borreliae) were used for enzymatic amplification. When 1/20 or 1/10 of the PCR-amplified products were used either for dot blot or Southern blot hybridization, the accessible copies of amplified B. burgdorferi DNA were sufficient for detectable hybridization to occur. PCR amplification of B. burgdorferi DNA in clinical specimens followed by dot blot hybridization may be a valuable adjunct or alternative to current but inadequate laboratory methods for the diagnosis of Lyme disease.