TCRAV segments contribute significantly to MHC restriction as illustrated by their general preference for either the CD4 or CD8 T cell subset and additional, MHC allele-specific overselection during T cell differentiation. The 10-fold over-representation of the TCRAV8S2 (VA8S2) segment in CD8 over CD4 T cells by the RT1(f) haplotype of LEW.1F rats provides the most striking example of MHC allele-specific overselection of a VA segment reported so far. Also in alloreactivity, VA8S2(+) CD8 cells from RT1(f-) rats are preferentially expanded by RT1(f+) stimulators. We have identified the class I molecule, A(f), mediating VA8S2 overselection and report that it differs only in four amino acids at the MHC-TCR interface from the class I molecule A(a), which is neutral with regard to selection of VA8S2. We also provide an extensive survey of the TCRAV8 family and show that among 14 functional VA8 segments in LEW rats, the dramatic A(f)-dependent overselection is unique for VA8S2. Surprisingly, VA8S2 expression in CD8 T cells of RT1(f+) rats derived from a Sprague-Dawley stock was only 3% as compared to the 12% observed in LEW.1F. The VA8S2 segment of Sprague-Dawley (VA8S2(SD)) differs from VA8S2 of the LEW background (VA8S2(l)) in only two amino acids, one of which is located in CDR2 and could thus participate in allele-specific recognition of A(f). However, analysis of the pre- and postselection thymic repertoires of Sprague-Dawley and LEW.1F rats and of the repertoire of CD8 cells from both strains expanded in the alloreactive response to RT1(f) revealed that the difference in VA8S2 representation between the two backgrounds is explained by differential availability in the preselection repertoires and not by a difference in overselection. Sequence comparisons of A(f) and A(a) and of both VA8S2 segments suggest a predominant role of CDR1 in hyper-reactivity to A(f). Thus, the VA composition of the mature TCR repertoire is influenced by TCRA: locus polymorphisms at two levels: the regulation of VA usage in the preselection repertoire and the composition of structural elements which contribute to specific VA-MHC interactions during thymic selection.