Recently human cartilage gp-39 (HC gp-39) was identified as a candidate autoantigen in rheumatoid arthritis (RA). To further investigate the relevance of this Ag in RA, we have generated a set of five mAbs to a combination epitope of complexes of HC gp-39(263-275) and the RA-associated DR alpha beta 1*0401 HLA class II molecules. FACS studies revealed that these mAb recognize specific complexes on homozygous DR alpha beta 1*0401-positive B lymphoblastoid cells pulsed with HC gp-39(263-275). The best mAb, 12A, was further characterized using a set of irrelevant DR alpha beta 1*0401-binding peptides and truncated/elongated versions of HC gp-39(263-275) itself. The minimal epitope recognized in combination with DR alpha beta 1*0401 was HC gp-39(263-273). Peptides not encompassing HC gp-39(263-273) were not recognized. Three of five mAb were able to inhibit (up to 90%) the response of HC gp-39(263-275)-specific DR alpha beta 1*0401-restricted T cell hybridomas to peptide-pulsed APC or purified complexes. Using mAb 12A, we have been able to identify and localize dendritic cells that present DR alpha beta 1*0401/HC gp-39(263-275) complexes in synovial tissue of DR alpha beta 1*0401-positive RA patients, indicating local presentation of the HC gp-39(263-275) epitope in the inflamed target tissue by professional APC. These data support a role of HC gp-39 in the local autoimmune response that leads to chronic inflammation and joint destruction.