It has been reported that collagen II (CII)-derived peptide induced T-cell activation via its amino acids responsible for T-cell receptor (TCR) recognition. In this study, three altered CII263–272 peptide ligands (APL) containing multiple substitutions of TCR contact residues were synthesized. Their roles in inhibition of T-cell activation were evaluated in peripheral blood lymphocytes (PBL) of rheumatoid arthritis (RA) in vitro. It was shown that 41% (25/61) of RA patients were responsive to the wild-type antigenic CII263-272. In contrast, marginal or silent T-cell responses to the three APLs were found, accompanied by inhibitory effects on secretion of Th1 type cytokines and expression of cell surface markers, CD69 and CD25. In addition, T-cell activation induced by the wild-type antigenic CII263-272 was inhibited by all the three APLs in a dose-dependent manner. It is demonstrated that APLs with substitutions of TCR contact residues are capable of down-regulating T-cell responses in PBLs of RA, suggesting that the CII-derived APLs are potentially therapeutic in RA. D 2005 Elsevier Inc. All rights reserved.