The polymerase chain reaction (PCR) was developed for use in the detection of Borrelia burgdorferi sensu lato, the Lyme disease agent. A 333-bp fragment of the 30-kbp circular plasmid from Borrelia burgdorferi B31 was amplified and PCR products were analysed by DNA-DNA hybridization. Sensitivity was enhanced by addition of a carrier to the samples before treatment and enabled detection of as few as 1 to 10 bacteria. Specific products were obtained only with the Lyme disease agents, but not with other spirochetes or unrelated bacteria. B. burgdorferi sensu lato was detected in cerebrospinal fluid (CSF) from 11 out of 45 patients with confirmed Lyme neuroborreliosis. In a prospective study, 20 out of 315 CSF samples from potential patients were PCR-positive. Forty uninfected patients were PCR-negative.