The restricted usage of particular T cell receptor β chain genes in autoimmune disease was studied in LEW rats using T cell hybridomas specific for an immunodominant sequence of bovine retinal S-Ag, which induces experimental autoimmune uveoretinitis. T cell hybridomas from a pathogenic T cell line, R858, specific for residues 273-289 of bovine retinal S-Ag were analyzed in order to determine the contribution of their TCR Vβ to self specificity as determined by recognition of the pathogenic epitope represented in the autologous rat S-Ag sequence. Six different, functional TCR rearrangements were expressed by the panel of hybridomas, including two distinct Vβ8.2 rearrangements and functional Vβ10, Vβ14, Vβ19 rearrangements, and an unidentified Vβ gene. All hybridomas were Ag specific and reacted both to nonself-peptide derivatives as well as to self-peptide homologues. No unique pattern of peptide reactivity distinguished Vβ8.2+ hybridomas from Vβ8.2- hybridomas; all of the hybridomas were most reactive to the nonself sequences and reacted to self peptide with one to three orders of magnitude less sensitivity. However, all Vβ8.2+ hybridomas were much better responders overall and were activated by lower concentrations of all peptides than were Vβ8.2- hybridomas. Although Vβ8.2 gene usage is strongly associated with autoimmune pathology, these data show that in LEW rats several different TCR Vβ genes are utilized in response to a short pathogenic sequence of this autoantigen and show that Vβ8.2 receptors are not uniquely self-reactive. However, the enhanced reactivity to Ag of Vβ8.2+ hybridomas relative to Vβ8.2- hybridomas specific for the same peptide may help explain the close association of Vβ8.2 TCR gene usage with pathogenicity found in autoimmune disease models.