T cells recognize short linear peptides bound to major histocompatibility complex (MHC)-encoded molecules. Subtle molecular changes in peptide antigens produce altered peptide ligands (APL), which induce different T cell responses from those induced by the antigenic ligand. A molecular basis for how these slight molecular variations lead to such different consequences for the T cell has not been described. To address this issue, we have amino acid substitutions at the primary T cell receptor (TCR) contact residue of the murine hemoglobin determinant, Hb(64-76)/I-E(k) and produced 12 peptides that interact with the TCR of the T cell clone 3.L2. The 3.L2 T cell responds to these peptides, which vary 1 million-fold in their activity, and enables them to be ranked according to their relative ability to signal through the 3.L2 TCR. Such a ranking reveals that the ability of the 3.L2 T cell to respond to these peptides depends on how well the structure of the side chain at the primary TCR contact site mimics that of the Asn residue present in the antigenic ligand. These reactivity of the 3.L2 T cell also depends on an MHC contact residue that is next to the primary TCR contact residue, suggesting that conformation of the Asn side chain is important. By using nonnatural amino acids at a TCR contact residue, we have demonstrated that APLs can be rationally designed based on structure. These data are consistent with a model in which the affinity of a peptide-MHC complex for the TCR determines how the T cell will respond.