Incubation of AtT-20/D-1 mouse pituitary tumor cells with glucocorticoids leads to a progressive diminution in the number of cellular glucocorticoid receptors. Control incubations demonstrate that non-glucocorticoids don't cause this depletion and that the decrease is not due to deleterious alterations in the incubation medium or degradation of the steroid. Incubation of cells with 0.5 micrograms/ml of cycloheximide alone does not dramatically diminish receptor content. However, incubation of cells with both cycloheximide and dexamethasone results in receptor depletion, suggesting that depletion involves enhanced receptor degradation. The cell's ability to bind glucocorticoids can be regenerated by a 24- to 48-h incubation in steroid-free medium. However, unlike the depletion step, the repletion process is sensitive to cycloheximide.