An experimental investigation concerning the dissociation equilibrium in a gel was carried out by using Chelating Sepharose Fast Flow gel and buffer solutions. It was noted that the gel swelled or shrank somewhat to adsorb or release mobile ions and water, and the degree of change slightly depended on the pH of the solution. It was found that the relationship between the degree of dissociation of fixed two -CH2COOH groups in gel and the concentration of hydrogen ions in the outer aqueous phase could be correlated by an equation based on the dissociation equilibrium. It was shown that the relationships for dissociation equilibrium based on theory were closely consistent with the experimental values. Finally the total concentration of the fixed two -CH2COOH groups in gel was obtained as 2.68 x 10(-2) mol/kg-wet gel.