A CD4+ human T cell clone YN5-32 recognized a streptococcal M12p54-68 peptide in the context of HLA-DR4 and produced a large amount of IFN-gamma. We investigated responses of YN5-32 to 156 independent analogue peptides carrying single residue substitutions at residues 57 (position 1 (p1)) to 65 (p9) of the peptide. Approximately 30% of analogues at either Leu57 (p1), Ala60 (p4), or Asn62 (p6) residues exhibited TCR agonism to stimulate various magnitudes of proliferative responses in the T cell clone, and analogues exhibiting TCR antagonism are rare in these three residues. In analogues at either Glu58 (p2), Gln59 (p3), Tyr61 (p5), or Glu63 (p7) residue, 30 to 50% exhibited TCR antagonism. About 10% of analogues at Glu58 (p2) or Tyr61 (p5) stimulated proliferative responses, while 30 to 50% of analogues at Gln59 (p3) or Glu63 (p7) did so. Some of these TCR antagonistic analogues carrying relatively conservative amino acid substitutions partially activated the T cells to induce large increases in size and expression levels of CD4, CD11a (LFA-1), CD28, CD49d (VLA-4), and CD95 (Fas), and small increases in CD25 and CD44 expressions on the cell surface. None of the partially activating antagonistic analogues induced IFN-gamma production or anergy in T cells. Analogues with replacements of acidic amino acids at either Leu64 (p8) or Ser65 (p9) residue had dominant negative effects on T cell proliferation. Thus, altered peptide ligands with single residue substitutions in the antigenic peptide frequently stimulated the human T cell clone, in at least three different ways to exhibit agonism, antagonism, and antagonism with partial activation. Frequencies of analogue peptides exhibiting these three different effects on the T cell clone differed depending on the residue of the peptide substituted. Altered T cell responses induced by analogue peptides of a T cell epitope provide a system to analyze activation signals mediated by TCR, and to manipulate T cell responses.