The determinants recognized by two I-Ak-restricted hen egg-white lysozyme-specific T cell hybridomas were differentiated with a series of truncated or substituted peptides. The 10mer 52-61 was the smallest peptide that was immunogenic for both T cells. This peptide differed by a single residue, Leu56, from the corresponding autologous lysozyme peptide, which was nonimmunogenic. The addition of amino acids to the amino terminus of 52-61 increased the immunogenicity of the peptides for 3A9 T cells and decreased the immunogenicity for 2A11 T cells. By deleting or diiodinating Tyr53, the resulting peptides were rendered totally nonimmunogenic. In contrast, the 3-NO2-Tyr derivative was fully immunogenic for the 3A9 cells but completely nonimmunogenic for the 2A11 cells. Thus, two different, but very similar, determinants were generated by the same HEL peptide and the I-Ak molecule.