A search for mineralocorticoid binding sites in neural tissue was carried out on the basis of competition by two mineralocorticoid antagonists (spironolactone and glycyrrhetinic acid), a mineralocorticoid agonist (9 alpha-fluorocortisol) and a pure glucocorticoid (RU 26988). The hippocampus was selected for these studies, in view of its preferential concentration of binding sites for [3H]-aldosterone (ALDO) and two glucocorticoids: [3H]-corticosterone (CORT) and [3H]-dexamethasone (DEX). Inhibition studies performed with 5 X 10(-4)-5 X 10(-9) M spironolactone and with 5 X 10(-4)-5 X 10(-8) M glycyrrhetinic acid, showed a dose-response reduction of [3H]-ALDO, [3H]-CORT and [3H]-DEX binding, implying that in brain these compounds did not behave as exclusive ALDO antagonists. Two concentrations of 9 alpha-fluorocortisol (5 X 10(-9) and 2.5 X 10(-8) M) preferentially displaced [3H]-ALDO in comparison to [3H]-DEX or [3H]-CORT. Relative binding affinity (RBA) of 9 alpha-fluorocortisol was also higher for the mineralocorticoid than for the glucocorticoids. RU 26988 (5 X 10(-6)-2.5 X 10(-8)M) gave differential inhibition of the three ligands and its RBA for [3H]-DEX site was twice as high as for [3H]-CORT, and three orders of magnitude higher than for [3H]-ALDO binding sites, thus clearly separating sites for gluco- and mineralocorticoids. Further evidence for the presence of separate binding molecules for mineralo- and glucocorticoids in hippocampal cytosol was provided by ultracentrifugation on 16-41% glycerol gradients containing molybdate, which yielded sedimentation values of 9.88 +/- 0.27 for [3H]-DEX (n = 5), 10.48 +/- 0.27 for [3H]-CORT (n = 9) and 11.3 +/- 0.13 for [3H]-ALDO (n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)