The binding sites for aldosterone and a potent aldosterone antagonist (SC-26304) were studied in kidney cytosol from adienalectomized rats. Preformed cytosol and kidney slices were incubated with 3H-labeled steroids in a wide range of concentrations. The recovery and characteristics of the binding sites were affected by the incubation and homogenization conditions. High-affinity, Type I mineralocorticoid binding was reduced by more than 95% when cytosol was incubated at 25°C in the presence of calcium. Tissue dilution also affected the binding sites. SC-26304 was bound to high- and low-affinity receptors, similar to the binding of aldosterone. The physiologic response to aldosterone could result from binding to either or both sets of sites. Some of the physiologic responses to spirolactones could represent antagonism of the binding of aldosterone to either or both sites. A convenient method is presented for describing the relative occupancy of several different sites by any particular steroid. © 1978.