We report the study of one CD4+ T-cell clone that recognizes peptide HA306-320 in the context of autologous DR1101 molecules as well as of allogeneic DR1301, DR0402, DR1501, and DR1601 molecules. This degenerate T-cell recognition is mediated by a single T-cell receptor (TCR) as judged by both TCR-V beta sequencing and cold-target competition assays. Restriction analysis shows that substitutions of DR residues within the third hypervariable region result in a loss of T-cell reactivity, which is restored by additional substitutions in the first and/or second hypervariable regions. Thus, there is no correlation between antigen presentation abilities of the different allelic DR products and the degree of sequence homology between these products. DR residues whose substitution is compatible with T-cell recognition potentially interact with peptides rather than with TCRs by virtue of their location in the floor of the groove or as previously documented for residues of the alpha-helix. Furthermore, antigen presentation by allogeneic DR molecules occurs independently of their affinity for the peptide, as determined in cell surface-binding assays using biotinylated HA306-320. Altogether these data suggest that degenerate T-cell recognition mainly depends on an influence of polymorphic DR residues on the configuration adopted by the peptide in the DR groove so that the epitope is left intact.