The relative contributions of putative T-cell receptor (TCR)-contacting and peptide-binding residues of a major histocompatibility complex (MHC) class II restriction element to serologic and antigen-specific T-cell recognition were investigated by site-specific mutagenesis. Amino acids 70 and 71 in the DRβ1 domain cf DR4 Dw 10 are uniquely different from the other Dw subtypes of DR4. Residues 70 is predicted to be located at the membrane-distal surface of the class II molecule, where it may influence T-cell recognition by a direct interaction with a TCR. Residue 71 is predicted to form part of the antigen-binding groove where its influence on T-cell recognition may be mediated indirectly via an effect on peptide binding. Trsnfected murine L cells were produced expressing the products of DR4 Dw10B genes in which the codons for residues 70 and 71 had been mutated towards DR4 Dw14. Support for the predicted orientations of β-chain residues 70 and 71 was lent by the observation that only 70 plays an important role in the formation of a serologic determinant. Mutation of this residue was sufficient to produce recovery of recognition by a human monoclonal antibody, NI, which has specificity for all the DR4 subtypes with the exception of DR4 Dw10. The human T-cell clone HA1.7, specific for influenza virus hemagglutinin (HA) peptide 307-319 and restricted by DR1 Dw1, exhibits degeneracy of MHC restriction on the Dr4 Dw subtypes with the exception DR4 DW10. Alteration of DR4 Dw10 β-chain residues 70 or 71 alone failed to rescue antigen recognition by HA1.7; only L cells expressing DR4 Dw10 with both amino acid substitutions served as effective antigen-presenting cells, implying a key role for both residues in antigen-specific T-cell recognition. The doubly substituted form of Dr4 Dw10 was a more efficient restriction element for HA1.7 than DR4 Dw14 and DR1 Dw1. This implies that the conservative change from leucine (DR1 Dw1 and all the DR4 subtypes except Dw10) to isoleucine (DR4 Dw10) leads to the equivalent of a hetoroclitic response.