A polymerase chain reaction (PCR) assay that detects Borrelia burgdorferi DNA in cerebrospinal fluid (CSF) was evaluated as a diagnostic test for acute or chronic Lyme neuroborreliosis. In one laboratory, 102 samples were tested blindly, and 40 samples were retested in a second laboratory. In the first laboratory, B. burgdorferi DNA was detected in CSF samples in 6 (38%) of 16 patients with acute neuroborreliosis, 11 (25%) of 44 with chronic neuroborreliosis, and none of 42 samples from patients with other illnesses. There was a significant correlation between PCR results and the duration of previous intravenous antibiotic therapy. The overall frequency of positive results was similar in the second laboratory, but concordance between the laboratories and among primer-probe sets was limited because many samples were positive with only one primer-probe set. Thus, PCR testing can sometimes detect B. burgdorferi DNA in CSF in patients with acute or chronic neuroborreliosis, but with current methods, the sensitivity of the test is limited.