Autoreactive T cells represent a natural repertoire of T cells in both diseased patients and healthy individuals. The mechanisms regulating the function of these autoreactive T cells are still unknown. Ob1A12 is a myelin basic protein (MBP)-reactive Th cell clone derived from a patient with relapsing-remitting multiple sclerosis. Mice transgenic for this human TCR and DRA and DRB1* 1501 chains develop spontaneous experimental autoimmune encephalomyelitis. The reactivity of Ob1A12 is reported to be restricted to recognition of MBP peptide 85–99 in the context of DRB1* 1501. DRA/DRB1* 1501 and the patient’s other restriction element, DRA/DRB1* 0401, differ significantly in their amino acid sequences. In this study we describe an altered peptide ligand derived from MBP 85–99 with a single amino acid substitution at position 88 (Val to Lys; 88V→ K), that could stimulate the Ob1A12. TCR in the context of both DRA/DRB1* 1501 and DRA/DRB1* 0401. Analysis of a panel of transfected T cell hybridomas expressing Ob1A12. TCR and CD4 indicated that Ob1A12. TCR cross-reactivity in the context of DRA/DRB1* 0401 is critically dependent on the presence of the CD4 coreceptor. Furthermore, we found that activation of Ob1A12. TCR with MBP altered peptide ligand 85–99 88V→ K presented by DRB1* 1501 or DRB1* 0401 resulted in significant differences in TCRζ phosphorylation. Our data indicate that injection of altered peptide ligand into patients heterozygous for MHC class II molecules may result in unexpected cross-reactivities, leading to activation of autoreactive T cells.