The encephalitogenic potential of a segment of myelin basic protein in experimental autoimmune encephalomyelitis is not always mirrored by the ability of the peptide to mediate in vitro activation of encephalitogenic T cells. Recent studies from our laboratory have demonstrated that the responsiveness of Ag-specific T cells in experimental autoimmune encephalomyelitis is determined not exclusively by Ag but also by the nature of the APC. By varying APC during the in vitro selection of T cells, we could generate distinct sets of rat encephalitogenic T cells, as evidenced by the diversity of TCR usage. Here we establish the importance of APC in the activation of rat encephalitogenic T cells by myelin basic protein peptides. Peptides 69-84-Gly and (P80)68-86, which lacked stimulatory activity toward many encephalitogenic T cells in our proliferation assay when standard APC were used, become strongly stimulatory in the presence of less commonly used APC, i.e., an Ia+ T cell clone (LOA) or an Ia-inducible rat glial cell clone (F10). Nonstimulatory APC failed to activate encephalitogenic T cells even when major cytokines were added, suggesting that these cytokines are not among the factors limiting the activating potential of the APC. Thus, whether or not an immunocompetent T cell can be activated by a given Ag in an autoimmune response may be determined by the properties of APC. This finding has implications for current research efforts to identify pathogenic self proteins.