Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease that serves as a model of human intraocular inflammatory disease (uveitis) and is initiated in susceptible animals by immunization with retinal Ag, such as interphotoreceptor retinoid binding protein (IRBP) and S-Ag (SAg). Previous studies of TCR usage by uveitogenic T cells suggested a possible connection between pathogenicity of T lymphocytes and usage of V beta 8 family genes. Here, we have analyzed the T cell repertoire at the autoimmune site by examining V beta gene expression in the retinas of rats with SAg- or IRBP-induced uveitis. Our data show the following: 1) T cell response to SAg or IRBP is clonally heterogeneous: at least 15 of the 20 known rat TCR families were detected in the retinas of rats with EAU. 2) V beta 8+T cells were selectively increased in retina during the early stage of EAU, and the pattern of V beta 8 subfamily member utilization in the retina was Ag-dependent: V beta 8.2+ and V beta 8.3+ T cells were found in the retina of rats immunized with IRBP but only V beta 8.2+ cells in SAg-induced EAU. V beta 8.1 cells were not detected in any of the samples. 3) Kinetic change in the proportions of V beta 8.2+ and V beta 8.3+ T cells was observed in IRBP-EAU: on the day of clinical onset of disease, V beta 8.3+ cells were the only member of the V beta 8 family detected, but 24 h later, V beta 8.2+ T cells appeared and became the dominant V beta 8 clonotype in the retina. 4) The pattern of V beta 8 family member usage by T cells in uveitogenic lines specific to the corresponding Ag was similar to their utilization in the uveitic response in the retina. DNA sequence analysis of 75 V beta 8 cDNA clones from uveitogenic T cell lines revealed that, of 20 V beta 8 clones isolated from SAg-specific T cell lines, all were V beta 8.2 TCR, whereas among 55 V beta 8 clones from IRBP-specific lines, 36 were V beta 8.2 and 19 were V beta 8.3. In similarity to the retina, no V beta 8.1+ T cells were detected from these T cell lines. Taken together, our data suggest a bias towards usage of V beta 8+ cells in EAU and, depending on the autoantigen mediating the disease, particular V beta 8 subfamily member(s) were preferentially utilized or excluded from the autoimmune response.