To discover novel autoantigens associated with Lyme arthritis (LA), we identified T-cell epitopes presented in vivo by human leukocyte antigen (HLA)-DR molecules in patients' inflamed synovial tissue or joint fluid and tested each epitope for autoreactivity. Using this approach, we identified the highly expressed human protein, apolipoprotein B-100 (apoB-100), as a target of T- and B-cell responses in a subgroup of LA patients. Additionally, the joint fluid of these patients had markedly elevated levels of apoB-100 protein, which may contribute to its autoantigenicity. In patients with antibiotic-refractory LA, the magnitude of apoB-100 antibody responses correlated with increased numbers of plasma cells in synovial tissue, greater numbers and activation of endothelial cells, and more synovial fibroblast proliferation. Thus, a subset of LA patients have high levels of apoB-100 in their joints and autoreactive T- and B-cell responses to the protein, which likely contributes to pathogenic autoimmunity in patients with antibiotic-refractory LA.