In this report, we investigate the activation of Th1 clones using altered TCR ligand. By changing the immunogenic peptide, cytolytic function can be separated from proliferative and lymphokine responses. These three responses were examined and dissected in two Th1 clones using analogs of the murine hemoglobin [Hb(64-76)] peptide. This analysis was focused on amino acids in the immunogenic peptide that were possible T cell contact residues. Typically, several amino acids were identified as critical contact residues for a Th1 proliferative response. An examination of lymphokine production (IFN-gamma or IL-3) revealed the same pattern of response to the analog peptides indicating that the proliferative and lymphokine responses were directly related. However, for cytolysis, fewer amino acid residues were identified as critical contact residues for effector function. Thus, some altered peptide ligands allowed the disassociation of the cytolytic function from the proliferative and lymphokine responses in Th1 clones. To extend these findings, the activation of T cell hybridomas created from the Th1 clones were similarly examined using the altered TCR ligands. The lymphokine response (IL-2) of the T cell hybridomas identified the same critical amino acids as did the cytolytic response of the Th1 clones. Thus, analog peptides partially activated the Th1 clones such that cytolysis occurred independent from proliferative and lymphokine responses.