HeLa S3 cells contain high affinity, saturable receptors specific for glucocorticoids (congruent to 20,000 per cell). Growth of HeLa S3 cells in media containing dexamethasone (10(-6) M) results in a pronounced (congruent to 70%) reduction in the total cellular level of nuclear or cytoplasmic dexamethasone receptor number without any alteration in steroid-receptor dissociation constant (Kd congruent to 1 X 10(-9) M). This reduction in receptor number is not the result of simple receptor occupation, since this effect also occurs when receptor number is modulated by the direct addition of [3H]dexamethasone. Both control and dexamethasone-treated cells attain equilibrium states of receptor binding by 120 min at 0 C and by 60 min at 37 C, enabling estimation of receptor number and affinity by Scatchard analysis of saturation curves. Down-regulation of glucocorticoid receptor occurs at steroid concentrations as low as 10(-9) M (40% reduction). This alteration in receptor occurs after 24 h of hormone but not after either 2 or 6 h. Only the active glucocorticoids, (dexamethasone, cortisol) down-regulate glucocorticoid receptors. Cortexolone, estradiol, and 5 alpha-dihydrotestosterone have no apparent effect on this process, whereas progesterone (10(-7) M) is partially effective. Subcellular distribution studies indicate that glucocorticoids affect only that population of receptors capable of nuclear translocation at 37 C. We conclude that glucocorticoids can regulate the metabolism of their own receptors, via undefined mechanisms.