Six of seven patients with Lyme arthritis were positive by PCR. In contrast, all 18 synovial fluid samples from patients with other disorders, including rheumatoid arthritis, spondyloarthropathy, gout, pseudogout, hemarthrosis, degenerative joint disease, lupus, papillary synovitis, and trauma, were negative by PCR (P < 0.001, Lyme arthritis compared with controls, Fisher exact test). All 38 laboratory controls were negative by PCR. The assay reproducibly detected 20 or fewer B. burgdorferi cells directly or when added to extracted synovial fluid that was previously negative by PCR. Polymerase chain reaction was done four times with identical results, including analyses with both outer surface protein A primer sets.