Primary cultures of mouse renal tubular epithelial cells were used to study the effect of hydrocortisone on the regulation of glucocorticoid receptors (GR) and on sodium-potassium adenosine triphosphatase (Na-K ATPase) activity. A GR assay was developed and performed directly on cell monolayers maintained in serum-free medium to which hydrocortisone at 5 nM, 50 nM, and 5 X 10(-4) M was added. Compared with control cells grown in medium without hydrocortisone, GR levels per cell decreased by 50% after 48 hours of growth in medium containing 5 nM hydrocortisone concentrations (50 nM or 5 X 10(-4) M), GR levels decreased to less than or equal to 28% of control values. In all hydrocortisone treatment groups there was an inverse relation between GR concentrations and Na-K ATPase activity. Binding of cell GR by the addition of the antiglucocorticoid RU 38486 in hydrocortisone-supplemented medium eliminated the glucocorticoid-induced stimulation of Na-K ATPase activity. These results demonstrate a time- and dose-dependent effect of glucocorticoids on GR binding activity and a direct relation between this receptor-hormone interaction and Na-K ATPase activity in intact renal tubular epithelial cells.