Although indirect evidence suggests that chronic Lyme arthritis is caused by persistent infection with Borrelia burgdorferi, direct visualization has been lacking. We report the demonstration of B. burgdorferi from synovial fluid aspirated from the right knee of a 31-year-old man with Lyme arthritis for more than 1 year. After 6 days, culture medium inoculated with synovial fluid showed one motile and several nonmotile spirochetes. Direct immunofluorescence staining showed reactivity with anti-B. burgdorferi serum. Spirochetes were not seen in subcultured material. The patient's arthritis improved with high-dose intravenous penicillin. Identification of B. burgdorferi from the joint fluid of a patient with long-standing arthritis supports the concept that the arthritis is due to persistent infection.