Pathogenic autoimmunity against oligodendrocyte-specific protein (OSP/claudin-11), recently implicated in multiple sclerosis (MS) pathophysiology, has been poorly investigated as compared to that against other myelin encephalitogens. Using recombinant soluble mouse OSP (smOSP) and overlapping peptides thereof, we show that smOSP-induced chronic EAE in C57BL/6J mice is primarily associated with CD4(+) T cells reactive against OSP179-207 and OSP22-46, the major and minor encephalitogenic regions, respectively, and with a predominant B cell response against OSP22-46. The encephalitogenic OSP179-207-specific T cells recognized OSP190-202 as minimal stimulatory epitope, while minimal encephalitogenic sequence was OSP191-199. Further delineation and structural bioinformatic analysis of the major encephalitogenic region suggested four overlapping potential I-A(b) core epitopes, predicting OSP192Y as major TCR-contact residue shared by OSP 188-196, OSP190-198, and OSP191-199 cores, albeit at different MHC-II pockets. Accordingly, substitution at OSP192Y yielded OSP188-192A-202, a non-stimulatory/non-encephalitogenic altered peptide ligand (APL) that was antagonistic for OSP188-202-specific encephalitogenic T cells. Systemic administration of OSP188-192A-202 suppressed OSP188-202-induced EAE and fully reversed smOSP-induced EAE. These data suggest that a single epitopic residue (OSP192Y) governs the selection and control of most pathogenic T cells associated with smOSP-induced EAE in H-2(b) mice. This may impact profoundly on peripheral self-tolerance to OSP and on potential APL-mediated therapy of OSP-related autoimmune pathogenesis.