3H-Aldosterone-binding proteins (ABP) were isolated from nuclear fractions of rat kidney, duodenal mucosa, spleen, liver and brain by extraction in Tris-CaCI2 and precipitation in 50% (NH4)2SO4. Cytosol (105,000 × g supernatants) ABP were isolated from the same tissues by G-50 Sephadex chromatography. The ABP content of the nuclear fractions was in the order: kidney ≈duodenal mucosa > spleen ≈liver≈ brain (table I). The ABP content of the cytosol fractions was in the order: kidney≈duodenal mucosa > spleen ≈ brain≈liver (table III). The relative affinities of the competing steroids, cold d-aldosterone, 9α-fluorocortisol, 17-isoaldosterone and 17/S-estradiol for the 3H-aldosterone binding sites were similar in both the nuclear and cytosol fractions of kidney, duodenal mucosa, spleen and liver. The active mineralocorticoids competed successfully for the 3H-aldosterone binding sites but the inactive steroids were either without effect or tended to enhance the binding of 3H-aldosterone. The physiological significance of relatively low levels of stereospecific ABP in spleen, liver and brain remains to be elucidated.