Aldosterone binds to two renal cytosol receptors, one with high affinity and low capacity (Type I or mineralocorticoid) and another with lower affinity but greater capacity (Type II or glucocorticoid receptor). One of the ways to study Type I receptors isolated from Type II receptors is to block the latter with a steroid which shows high affinity for the glucocorticoid receptor and low affinity for the mineralocorticoid receptor. Dexamethasone has been used widely for this purpose but previous investigations and this study have found that dexamethasone competes with [3H]aldosterone for Type I receptor binding with a relative activity of 26% that of aldosterone and therefore is less than ideal as a glucocorticoid receptor blocker. RU-26988 (11 beta, 17 beta-dihydroxy-17 alpha-pregnane-1,4,6-trien-20-yn-21-methyl-3-one) is a recently synthesized glucocorticoid that shows very low affinity for the mineralocorticoid receptor. RU-26988 competes with [3H]aldosterone and [3H]2 alpha-methyl-9 alpha-fluorocortisol, a powerful mineralocorticoid, for the cytosol receptor from renal slices of adrenalectomized rats with a relative potency of less than 0.5% in comparison to unlabeled aldosterone and 2 alpha-methyl-9 alpha-fluorocortisol. RU-26988 has over twice the ability of unlabeled dexamethasone to compete with [3H]dexamethasone for binding to the renal cytosol glucocorticoid receptor. Scatchard analysis of [3H]aldosterone binding to rat renal cytosol showed two receptors, one with a calculated dissociation constant (Kd) of 2.81 X 10(-9) M and a second with a calculated Kd of 3.33 X 10(-8) M. The addition of a 100-fold concentration of RU-26988 produced a single line with a Kd of 5.02 X 10(-10) M indicating that the specific blocking of the glucocorticoid receptors allows an accurate determination of the kinetic parameters of the mineralocorticoid receptor by itself. Scatchard analysis of [3H]2 alpha-methyl-9 alpha-fluorocortisol binding produced a straight line with a Kd of 3.7 X 10(-9) M, such as would be produced if it were binding to only one single class of receptors. However, when an excess of RU-26988 was added to block the glucocorticoid receptor, a different straight line was produced by Scatchard's analysis with a Kd of 3.78 X 10(-10) M. Whereas the explanation for this is not apparent, it may be that the much larger concentration of glucocorticoid receptors, for which 2 alpha-methyl-9 alpha-fluorocortisol also has a very great affinity, masks the binding to the mineralocorticoid receptors.