Several observations in the characterization of EAU are examined. First, sequences of heterologous S-Ag (bovine S-Ag in LEW rats) which induce strong in vitro T cell proliferative responses are dissociated from sequences which induce EAU. Strong in vitro responses were detected only to nonself peptide homologues. Second, T cells specific for self-sequences of S-Ag are unresponsive in vitro. Third, TCR Vβ8 gene usage is associated with pathogenic T cells. Vβ8.2 bearing hybridomas from a pathogenic line exhibited enhanced reactivity to pathogenic self-peptides, but were unresponsive unless presented Ag on nonirradiated, splenic APC. We propose that these findings reflect self, nonself discrimination of the epitopes on heterologous autoantigen, and examine the hypothesis that TCR containing Vβ8 have enhanced avidity for MHC complexed with autologous sequences, but that these Vβ8 autoreactive T cells appear unresponsive in vitro due to mechanisms of self-tolerance involving superantigen/coligand participation.