It has been proposed that the generally low affinities of T cell receptors (TCRs) for their peptide-major histocompatibility complex (pMHC) ligands (Kd approximately 10(-4) to 10(-7) M) are the result of biological selection rather than an intrinsic affinity limitation imposed by the TCR framework. Using a soluble version of the 2C TCR, we have used complementarity determining region (CDR)-directed mutagenesis to investigate whether the affinity of this receptor for its allogeneic pMHC ligand can be improved upon. We report that several mutants at positions lying within CDR3alpha and CDR2beta showed increased affinities for pMHC compared with the wild-type receptor. Additionally, we have investigated whether Valpha mutations that have been implicated in the phenomenon of CD8(+) repertoire skewing achieve this skewing by means of generalized increases in affinity for MHC-I molecules. Two mutants (S27F and S51P), which each promote skewing toward a CD8(+) phenotype, exhibited significantly reduced affinity for pMHC-I, consistent with a quantitative-instructional model of CD4/CD8 lineage commitment. This model predicts that CD8 is downregulated on thymocytes that have TCR-ligand interactions above a minimal energy threshold. Together, the results (a) demonstrate that engineering higher affinity TCRs is feasible, and (b) provide TCR-pMHC energy values associated with CD4/CD8 repertoire skewing.