The mineralcorticoid receptor in cytosol from the kidneys and hippocampi of adrenalectomized rats was characterized and quantified by use of isoelectric focusing in slabs of polyacrylamide gel. The mineralcorticoid receptor was labeled with [3H]aldosterone after selective blockade of the glucocorticoid receptor sites with a synthetic pure glucocorticoid RU 26988 [11 beta, 17 beta-dihydroxy-17 alpha-(1-propionyl)androsta-1,4,6-trien-3-one]. The apparent dissociation constant for aldosterone was 5.1 X 10(-9) M, and the ligand specificity studies revealed a mineralcorticoid pattern. The glucocorticoid and mineralocorticoid receptors were compared by limited trypsin digestion, followed by isoelectric focusing. Completely different fragmentation patterns were obtained for the two receptors in the same cytosol from either kidney or hippocampus. However, comparison of the mineralcorticoid receptor from the kidney with that from the hippocampus gave rise to identical fragmentation patterns. An antiglucocorticoid receptor antibody showed no crossreactivity with the mineralcorticoid receptor. Cytosol from kidney and hippocampus contained 28 +/- 24 and 155 +/- 115 fmol mineralcorticoid receptors/mg cytosol protein, respectively. Significant amounts of RU 26988-resistant [3H]aldosterone-binding sites were also present in cerebrum, cerebellum, spleen, and colon.