Immunization of mice with two myasthenogenic peptides, p195–212 and p259–271, which are sequences of the human acetylcholine receptor, resulted in myasthenia gravis (MG)-associated immune responses. A dual altered peptide ligand (APL) composed of the two APLs of the myasthenogenic peptides inhibited, in vitro and in vivo, those responses. The aims of this study were to further elucidate the mechanism/s by which the dual APL down-regulates MG-associated responses in vivo and characterize the cell population/s involved in this immunomodulatory suppressive effect. We have shown here that s.c. administration of the dual APL activates CD4CD25-expressing cells in lymph nodes (LN) of SJL mice. Furthermore, depletion of these cells diminished significantly the inhibitory effect of the APL on p195–212-specific proliferative responses. Depletion of the CD4⁺CD25⁺ cells was accompanied with a decrease in the secretion of the immunosuppressive cytokine, transforming growth factor (TGF)-β. Administration of the dual APL resulted also in the up-regulation of the expression of cytotoxic T lymphocyte antigen (CTLA)-4 and in a down-regulated expression of CD28 on LN cells. Blockade of the CTLA-4 function, in vitro, abrogated the inhibitory effect of the dual APL on the proliferative responses specific to p195–212. Thus, our results suggest that the active suppression exerted by the dual APL is mediated by the CD4⁺CD25⁺ immunoregulatory cell population, either directly through the CTLA-4 molecule expressed on these cells, and/or indirectly by causing the differentiation of other regulatory T cell population/s that secrete immunosuppressive cytokines.