A pronounced decrease in specific dexamethasone binding capacity of heart cytosol was observed. The inactivation of unbound glucocorticoid receptor was not due to the action of proteases since the rat of albumin digestion was very slow in the particulate preparations. The rate of inactivation of glucocorticoid binding capacity depends on cytosol protein concentration and temperature. Addition of hormone and glycerol to cytosol markedly slow binding capacity inactivation. The rapid loss of binding activity, especially in the cytosol with high protein concentration, makes difficult to perform equilibrium studies for simultaneous determination of binding affinity and a number of binding sites. Under various conditions the apparent dissociation constants were measured and compared with the dissociation constants, calculated from the rate of constants of association and dissociation, in order to find out optimal conditions for simultaneous determination of KD and [R0].