TCR V alpha elements are expressed preferentially in CD4 or CD8 subsets in a manner that is largely independent of MHC haplotype. It is likely that the V alphas interact preferentially with conserved regions of class I or class II molecules. To investigate the topology of binding of TCR to MHC-peptide complexes, we screened a panel of H-2Kbm mutants for differential V alpha expression. One strain, bm23, showed a consistent alteration in V alpha expression, with increased V alpha 3.2 expression in CD8 peripheral T cells. This overselection is manifest in CD8 single-positive thymocytes and appears to be due to enhanced positive selection on Kbm23. There is no apparent effect of V beta elements. The Kbm23 molecule is unique compared with Kb and the other Kbm molecules at residue 75 on the helix of the alpha 1 domain, suggesting an interaction between V alpha 3.2 and the alpha 1 helix at this point. Such an interaction is inconsistent with the orientation of TCR and MHC defined in two crystal structures, but is consistent with an orientation where the TCR is rotated by 180 degrees relative to MHC.