Using a primary limiting dilution approach to generate T cell lines, we compared myelin basic protein (MBP)-specific T cell clones from naive unprimed Lewis rat thymuses with the corresponding T cell repertoire of primed rats. We found that in the native thymus repertoire MBP-specific, encephalitogenic T cell clones preferentially use T cell receptor V beta 8.2 genes, along with CDR3 sequences typical for the primed Lewis anti-MBP response. In contrast to T cells from primed immune organs, which all display the CD4+CD8- phenotype, the majority of naive thymus-derived T cell clones expressed reduced levels of the CD4 co-receptor. Some clones were completely CD4-CD8-, while others included CD4-CD8- subpopulations along with CD4+CD8- T cells. In the one mixed population examined in detail, the CD4-CD8- and CD4+CD8-T cell subpopulations used a T cell receptor with identical beta chain sequence. The data suggest that in the Lewis rat the biased T cell receptor gene usage by encephalitogenic T cells is a property of the natural thymic T cell repertoire, possibly as a consequence of positive selection. The unusually low expression of CD4 in the major histocompatibility complex class II-restricted autoreactive T cells could be related to their escape from negative selection within the thymus.