The behavior of mineralocorticoid and glucocorticoid receptors of rabbit kidney cytosol was investigated on two affinity gels: a new affinity matrix prepared with a 3-O-derivative of carboxymethyloxime deoxycorticosterone (deoxycorticosterone gel) and a gel linked to a 17 beta-dexamethasone derivative (dexamethasone gel). Deoxycorticosterone gel was highly specific, since it retained mineralocorticoid but not glucocorticoid receptors, and dexamethasone gel exhibited high selectivity for glucocorticoid receptors since it did not bind mineralocorticoid receptors. The use of these two matrices allowed separation of mineralocorticoid and glucocorticoid receptors and further characterization of each type of cytosolic receptors after its isolation. Cytosolic mineralocorticoid and glucocorticoid receptors stabilized by tungstate were found to have a Stokes radius of approximately 6 nm, as determined by high performance size exclusion chromatography and a sedimentation coefficient of approximately 9 S, determined on a glycerol density gradient containing tungstate, under either high or low salt conditions. The hydrodynamic parameters, binding characteristics, and specificity of mineralocorticoid receptors were the same in the untreated and dexamethasone gel-treated cytosol. Similarly glucocorticoid receptor characteristics remained unchanged after deoxycorticosterone gel treatment, indicating biochemical independence of cytosolic mineralocorticoid and glucocorticoid receptors. The [3H]aldosterone receptor complex eluted from deoxycorticosterone gel was recovered with a 30-40% yield and a purification factor of about 1000. Purified mineralocorticoid receptors had the same sedimentation coefficient as cytosolic mineralocorticoid receptors (9 S) but a different Stokes radius (4 versus 6 nm). The decrease in the Stokes radius of the purified mineralocorticoid receptors was probably due to the gel filtration method. These results indicate that the newly synthesized matrix specific for mineralocorticoid receptors constitutes a powerful tool for their extensive purification.