We have generated a panel of cross-reactive T cells by immunizing SJL mice (I-A(s)) with Q144 peptide, an analog of an autoantigenic peptide (W144) of myelin proteolipid protein (PLP) 139-151 (HSLGKWLGHPDKF) in which W was replaced by Q at position 144. Following immunization with Q144, T cells were expanded in vitro with W144, which is a cross-reactive, suboptimal ligand, for Q144-specific T cells. The T cell clones responded to both ligands and grew normally on the peptide W144, but were hyperstimulated when activated by Q144 in vitro. This hyperstimulation results in a heteroclitic proliferative response with secretion of additional cytokines not induced by W144. Thus expansion of T cells by a suboptimal cross-reactive ligand effectively lowers the activation threshold so that the immunizing antigen becomes a hyperstimulating ligand for the clones. Surprisingly, when the T cell clones are grown on the hyperstimulating ligand Q144, some adapt by increasing their activation threshold. This desensitization results in a loss of response to a number of cross-reactive ligands and the appearance of a more specific T cell response. Long-term culture with the hyperstimulating ligand is sometimes associated with down-regulation of CD4 expression. These results provide an explanation for the common finding of T cell heteroclicity, and suggest that although the specificity and hierarchy of the response of T cells to peptides is determined by the TCR, activation threshold and effector functions are modified by exposure to cross-reactive ligands. This observation has implications for the development and regulation of autoimmune disease.