Hydrocortisone (HC) in rat liver cytoplasmic fraction was bound to three different binding sites with high, medium and low affinity. Dissociation constants (Kd) were approx. 2.1, 22 and 208 nM; and the densities of these binding sites were about 40, 50 and 10% of total number of binding sites, respectively. The binding site for dexamethasone (DM) of the cytoplasmic fraction was the medium affinity one among these three components. The maximum number of binding sites (Bmax) of HC and DM was significantly increased by adrenalectomy. The Bmax of HC was about twice as great as that of DM in normal and adrenalectomized rat liver. DM inhibited 3H-HC binding in a dose-dependent manner but inhibition did not exceed 50% in either normal or adrenalectomized rats. Following adrenalectomy, the Bmax of the medium affinity-site for HC was significantly increased, while the high affinity component disappeared. By adding DM to the cytoplasmic fraction of adrenalectomized rat liver in vitro and in vivo, the Bmax of the medium affinity-site was significantly decreased, and a high affinity component of HC was revealed with a significant increase in the number of binding sites. These results indicate that the binding site for DM is one component of the HC binding site; and following adrenalectomy, the number of each type of binding site for glucocorticoids increases differently from the others.