Abstract T lymphocyte activation is mediated by the interaction of specific TCR with antigenic peptides bound to MHC molecules. Single amino acid substitutions are often capable of changing the effect of a peptide from stimulatory to antagonistic. Using surface plasmon resonance, we have analyzed the interaction between a complex consisting of variants of the MCC peptide bound to a mouse class II MHC (E k) and a specific TCR. Using both an improved direct binding method as well as a novel inhibition assay, we show that the affinities of three different antagonist peptide–E k complexes are ∼10–50 times lower than that of the wild-type MCC–E k complex for the TCR, largely due to an increased off-rate. These results suggest that the biological effects of peptide antagonists and partial agonists may be largely based on kinetic parameters.