Abstract A major feature of the TCR repertoire is strong alloreactivity. Peptides presented by allogeneic MHC are irrelevant for recognition by a subset of alloreactive T cells. To characterize peptide-independent TCRs at the molecular level, we forced the expression of a TCRβ chain isolated from a peptide-independent alloreactive CD8+ T cell line. The alloreactive TCR repertoire in the transgenic mouse was peptide dependent. However, analysis of essential TCR contacts formed during the recognition of self-MHC-restricted Ag showed that fewer contacts with peptide were established by the transgenic TCRβ chain, and that this was compensated by additional contacts formed by endogenous TCRα chains. Thus, reduced interaction with the peptide appears to be a transferable feature of the peptide-independent TCRβ chain. In addition, these findings demonstrate that reactivity to peptides is preferred over the reactivity to MHC during the formation of the TCR repertoire.