We examined the specificity of positive and negative selection by using transgenic mice carrying a variant of the D10 TCR. We demonstrate that a point mutation at position 51 within the CDR2α segment significantly reduces the avidity of this TCR for its cognate ligand, but does not impact recognition of nonself MHC class II molecules. Although structural studies have suggested that this TCR site interacts with the MHC class II β-chain, the avidity of this TCR for its ligand and the function of the T cell can be reconstituted by a point mutation in the bound antigenic peptide. These data demonstrate that the bound peptide can indirectly alter TCR interactions by influencing MHC structure. Remarkably, reducing the avidity of this TCR for a specific antigenic peptide-MHC ligand has a dramatic impact on thymic selection. Positive selection of thymocytes expressing this TCR is nearly completely blocked, whereas negative selection on allogenic MHC class II molecules remains intact. Therefore, the recognition of self that promotes positive selection of the D10 TCR is highly peptide-specific.