Three arginine residues of the binding site of the Escherichia coli aspartate receptor contribute to its high affinity for aspartate (K(d) approximately 3 microm). Site-directed mutations at residue 64 had the greatest effect on aspartate binding. No residue could substitute for the native arginine; all changes resulted in an apparent K(d) of approximately 35 mm. These mutations had little impact on maltose responses. At residue Arg-69, a lysine substitution was least disruptive, conferring an apparent K(d) of 0.3 mm for aspartate. Results obtained for an alanine mutant were similar to those with cysteine and histidine mutants (K(d) approximately 5 mm) indicating that side chain size was not an important factor here. Proline and aspartate caused more severe defects, presumably for reasons related to conformation and charge. The impact of residue 69 mutations on the maltose response was small. Mutations at Arg-73 had similar effects on aspartate binding (K(d) 0.3-7 mm) but more severe consequences for maltose responses. Larger side chains resulted in the best aspartate binding, implying steric considerations are important here. Signaling in the mutant proteins was surprisingly robust. Given aspartate binding, signaling occurred with essentially wild-type efficiency. These results were evaluated in the context of available structural data.