In previous studies we have found that intact AtT-20 cells contained two glucocorticoid binding sites with distinctly different affinities and specificity. In this paper, the nature of these sites was investigated by studying glucocorticoid binding to cytosol and to plasma membranes isolated from AtT-20 mouse pituitary tumor cells. Plasma membrane vesicles were isolated from AtT-20 cells and found to take up alpha-aminoisobutyric acid, indicating that they were properly oriented and functionally intact. Corticosterone bound to these vesicle in a time- and temperature-dependent manner. The binding exhibited a glucocorticoid preference since non-glucocorticoids such as progesterone, testosterone or estradiol were unable to inhibit binding. In addition, binding specificity differed from that of the cytoplasmic receptor since the synthetic glucocorticoids were also ineffective competitors. The major inhibitors of binding were corticosterone greater than 11-dehydrocorticosterone greater than 11-ketoprogesterone greater than cortisol. In other complementary studies, AtT-20 cell cytosol was tested to determine whether heterogenous soluble sites exhibiting a preference for the natural vs the synthetic steroid could also be identified. We found that binding sites for both steroid classes were approximately similar in number, specificity and behavior on ion-exchange chromatography. We conclude that, in addition to a classical soluble cytoplasmic glucocorticoid receptor, AtT-20 cells contain plasma membrane glucocorticoid binding sites. The affinity and specificity of these sites for the natural ligand, corticosterone, suggest that they play an important role in the subcellular mechanism of glucocorticoid action.