The existence of high and low affinity mineralocorticoid-binding macromolecules (receptors) has been demonstrated in vitro in cytosols derived from the adrenalectomized rat brain by the specific binding of [3H]aldosterone (3H-A). The high-affinity aldosterone sites can be distinguished from those sites which have a higher affinity for either [3H]dexamethasone (3H-DM) or [3H]corticosterone (3H-B) on the basis of selectivity for spirolactone SC-9420 or non-radioactive A, DM, and B. The binding of 3H-A to the receptors was maximal after 2 hours of incubation of 0-4C. No significant binding of 3H-A to the receptors could be demonstrated when incubations of the radioactive ligand were performed at either 20 or 37 C, indicating that the receptor is heat-liabile. Scatchard analysis of the 3H-A binding data over a 200-fold concentration range of 3H-A indicated that there are two binding sites for aldosterone, a high affinity component (a1) with a Kd approximately equal to 1.5 X 10(-9)M and a low-affinity component (a2) with a Kd approximately equal to 6.3 X 10(-8)M. A similar study using 3H-DM as the radioactive ligand demonstrated only one site for the 3H-DM binding with a Kd = 6.2 X 10(-9)M. The presence of specific aldosterone receptors in the brain with high affinity, limited capacity, and selectivity for aldosterone suggests a possible extra-renal mechanism of action of the hormone in or mediated through the CNS.