The interaction between EMI (eosin-5-maleimide) and band 3 of human erythrocytes was studied under various conditions. It was found that the effects of the ionic strength on the EMI-band 3 interaction strongly depended on pH. At pH 6.0, the ionic strength had remarkable effects on the EMI-bound band 3, whereas at pH 7.4, the EMI-band 3 interaction was independent of ionic strength. From the change in the circular dichroism spectra of the EMI-bound band 3, it was revealed that the conformation or the structure of the EMI-binding sites in the cytoplasmic domain of band 3 was strongly dependent on ionic strength. The thermodynamic parameters for the covalent-binding between EMI and band 3 were calculated on the basis of the difference spectra of the EMI and ghost system. The values of activation energy and activation entropy change at pH 6.0 were extraordinarily small compared with those values at pH 7.4. These findings represent the characteristics of the EMI-binding sites in the cytoplasmic domain of band 3. The interaction of EMI with an isolated fragment of the cytoplasmic domain, 43k fragment, was also examined. The circular dichroism spectra of the EMI-bound 43k fragments was significantly different from those of the EMI-bound band 3. This may indicate that the quaternary structure of the EMI-binding site in the cytoplasmic domain of band 3 is altered by an allosteric connection with the membrane-spanning domain of band 3. Further, from the pH titration of the 43k fragment, it was suggested that lysine residues are responsible for the ionic interaction between EMI and the 43k fragment.