Cytosolic binding of [3H]dexamethasone was studied in the colon of the rat. [3H]dexamethasone binding was rapid and stable at 4 degrees C for 240 min. Scatchard analysis revealed a Kd of 6.2 +/- 0.5 X 10(-9) M and a binding capacity of 149 +/- 6.4 fmol/mg cytosolic protein (100,000-g fraction). Although dexamethasone inhibited [3H]dexamethasone binding more than that of aldosterone, [3H]aldosterone binding was inhibited equally by both aldosterone and dexamethasone. The relative order of potency of other steroids to inhibit [3H]dexamethasone binding was: dexamethasone greater than progesterone greater than spironolactone greater than aldosterone greater than corticosterone greater than cortexolone greater than estradiol. In other experiments, low doses of dexamethasone and aldosterone were infused into adrenalectomized animals to determine functional importance of these cytosolic steroid receptors. One-hour infusion of aldosterone at 2 micrograms/100 g body wt, which was the minimal dose of dexamethasone that increased transmural potential difference, did not alter the potential difference. These studies demonstrate the presence of a cytosolic receptor for dexamethasone and suggest that the action of dexamethasone on electrolyte transport in adrenalectomized animals is not mediated by the mineralocorticoid type of receptor and may be mediated by the specific high-affinity receptor for dexamethasone.