The use of altered peptide ligands (APL) with TCR antagonist properties holds promise as an antigen-specific therapy for autoimmune disorders. We are investigating the therapeutic potential of APL in experimental autoimmune encephalomyelitis (EAE) using the Ac1-9 peptide of myelin basic protein in H-2u mice. Encephalitogenic T cells recognize Ac1-9 using residues 3Gln and 6Pro as the major TCR contact sites. Use of position 6 APL is compromised by the heterogeneous nature of the Ac1-9-specific repertoire. Here we identify two position 3 APL that act as TCR antagonists on transgenic T cells expressing Ac1-9-specific TCR and that inhibit EAE in H-2u mice. However, the therapeutic capacity of these two APL correlated directly with the ability to maximally inhibit activation of a heterogeneous T cell pool. The implications of these findings for the requirements for EAE induction, the relative contribution of a given T cell subpopulation to pathology and the mechanism underlying EAE inhibition in this model are discussed.