The 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 TCR alpha chains, with a conserved junctional motif, but very different TCR beta chains. Modeling studies led to the prediction that the conserved glutamic acid residue in the TCR alpha chain could interact with the lysine at position 316 in the peptide, a known TCR contact residue. HA1.7 TCR-CD3 zeta chimeric constructs were expressed in the rat basophil line (RBL) and shown to confer specific antigen recognition. In two TCR alpha 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 TCR alpha chain paired with the HA 1.7 TCR beta 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.