Interaction of EDTA with horseradish peroxidase (HRP) was investigated by NMR relaxation-rate measurements. At pH 4.0, the apparent dissociation constant (Kd) for the EDTA binding to HRP was deduced to be 78 mM from the relaxation measurements. From pH-dependence of 1H-NMR line width of EDTA, it was observed that EDTA binds to HRP only under acidic conditions (pH < 5). The binding of EDTA to HRP was facilitated by protonation of an acid group on the enzyme with pKa 4.0. The Kd for EDTA binding to HRP was also evaluated in the presence of an excess of exogenous substrates such as iodide and thiocyanate ions. The Kd in the presence of iodide and thiocyanate ions showed that EDTA competes with these ions for the same binding site. The distance of EDTA protons from ferric centre of HRP were deduced from 1H-NMR relaxation measurements and was found to be in the order of 8 A.