The major amino acids necessary for diphtheria toxin (DT) binding to its receptor have been identified previously. Studies by W. H. Shen et al. (J. Biol. Chem. 269, 29077-29084, 1994) and by J. H. Cha ef al. (Mol. Microbiol. 29 (5), 1275-1284, 1998) suggested that the positively charged nature of the single amino acid residue, (516)Lys of DT, is crucial for binding to the DT receptor, whereas the negatively charged (141)Glu of the DT receptor is the most important residue for toxin binding. Here, we hypothesize that key interactions occur between these two oppositely charged amino acid residues. Reciprocal substitution of the residues at these positions between the toxin and the receptor was performed, which resulted in a partial reconstitution of the toxin:receptor interaction. This study provides the first biological data that characterizes the specific interaction of these two key residues with each other and also the additional interactions between other positively charged residues of DT and (141)Glu of the DT receptor.