Antigen-induced arthritis (AIA) in mice occurs after the single injection of methylated bovine serum albumin (mBSA) into the knee joint of animals preimmunized with the same antigen in complete Freund's adjuvant. A short acute reaction is followed by a chronic inflammation which shows similar histological features to human rheumatoid arthritis. The mechanisms leading to the chronicity of arthritis are not yet clear. Previous data suggest that autoimmune responses to cartilage components contribute to the persistence of arthritis. In the present study we estimate the frequency of autoreactive cells in the draining lymph nodes of arthritic mice by means of limiting dilution analysis. Graded concentrations of lymph node cells were stimulated for 7 days with type II collagen (CII) or cartilage proteoglycans (PG) in the presence of irradiated syngeneic feeder cells, and the frequency of responding cells was calculated. In the draining lymph nodes of immunized mice without arthritis induction, the frequency of CII reactive lymph node cells ranged from 1/41,182 to 1/57,424 whereas only 1 out of 3 experiments revealed a detectable frequency of PG reactive cells (1/136,128). For comparison, no CII or PG reactive cells were detected in the lymph nodes of normal mice. Intra-articular challenge of immunized mice with mBSA resulted in chronic destructive arthritis and caused a significant increase in the frequencies of CII and PG reactive lymph node cells (1/12,776 to 1/24,611 and 1/79,964 to 1/93,075, respectively). When using incomplete Freund's adjuvant for immunization, a comparable acute arthritis developed after the intra-articular injection of antigen, but the transition into the chronic stage was missing. The frequencies of autoreactive cells in the lymph nodes of these animals were below the level of detection and were judged to be below 1/150,000. The results suggest that autoimmune reactions against cartilage constituents might contribute to the perpetuation of joint inflammation, and that the mycobacteria in the complete Freund's adjuvant play an essential role in this process.