AbstractThe fine specificity of T cell receptor (TCR) interaction with the influenza hemagglutinin peptide HA 307–319 in the context of the DR1 (DRA, DRB1*0101) and DR4 (DRA, DRB1*0404) was studied in two human T cell clones (HA1.7 and Cl‐1) derived from different individuals. Sequencing of amplified TCR transcripts revealed that these two clones express highly related TCRα chains, with a conserved junctional motif, but very different TCRβ chains. Modeling studies led to the prediction that the conserved glutamic acid residue in the TCRα chain could interact with the lysine at position 316 in the peptide, a known TCR contact residue. HA1.7 TCR‐CD3ζ, chimeric constructs were expressed in the rat basophil line (RBL) and shown to confer specific antigen recognition. In two TCRα chain mutants, with the conserved glutamic acid residue altered to alanine and lysine, respectively, peptide recognition was lost. Specific recognition was not rescued by altered peptide ligands. Furthermore, Jurkat derivatives expressing the related Jurkat TCRα chain paired with the HA 1.7 TCRβ chain did not recognize the HA 307–319 /DR1 complex. These data provide evidence for the critical interaction of a TCR residue with antigenic peptide.