Susceptibility to multiple sclerosis (MS) is associated with the human histocompatibility leukocyte antigen (HLA)-DR2 haplotype, suggesting that major histocompatibility complex class II–restricted presentation of central nervous system–derived antigens is important in the disease process. Antibodies specific for defined HLA-DR2–peptide complexes may therefore be valuable tools for studying antigen presentation in MS. We have used phage display technology to select HLA-DR2–peptide-specific antibodies from HLA-DR2–transgenic mice immunized with HLA-DR2 molecules complexed with an immunodominant myelin basic protein (MBP) peptide (residues 85–99). Detailed characterization of one clone (MK16) demonstrated that both DR2 and the MBP peptide were required for recognition. Furthermore, MK16 labeled intra- and extracellular HLA-DR2–MBP peptide complexes when antigen-presenting cells (APCs) were pulsed with recombinant MBP. In addition, MK16 inhibited interleukin 2 secretion by two transfectants that expressed human MBP–specific T cell receptors. Analysis of the structural requirement for MK16 binding demonstrated that the two major HLA-DR2 anchor residues of MBP 85–99 and the COOH-terminal part of the peptide, in particular residues Val-96, Pro-98, and Arg-99, were important for binding. Based on these results, the antibody was used to determine if the HLA-DR2–MBP peptide complex is presented in MS lesions. The antibody stained APCs in MS lesions, in particular microglia/macrophages but also in some cases hypertrophic astrocytes. Staining of APCs was only observed in MS cases with the HLA-DR2 haplotype but not in cases that carried other haplotypes. These results demonstrate that HLA-DR2 molecules in MS lesions present a myelin-derived self-peptide and suggest that microglia/macrophages rather than astrocytes are the predominant APCs in these lesions.