The presence of sites specifically binding natural glucocortocoids in plasma membrane (PM) preparations (PM0, density=1.13-1.16; PM1, density=1.16-1.18) from rat liver was elucidated by equilibrium dialysis as well as by centrifugal methods. Equilibrium dialysis showed the presence of binding sites having a higher affinity for [3-H]cortisol (Kd=1.4 times 10- minus 9M at 4C) in PM0, and that of the binding sites having a lower affinity for [3H] cortisol (Kd=1.3 times 10- minus 8M at 4C) in PM1, while centrifugal analysis showed the presence of higher affinity binding sites (Kd=1.5-1.9 times 10- minus 97 at 0 C) in both PM0 and PM1, and also of intermediate affinity binding sites (Kd=4.1 times 10- minus 9M at 0 C) in PM1. The discrepancy in the cortisol binding parameters obtained by the two different methods seems to be due mainly to the lability of some binding sites, especially the PM1. The glucocorticoid-binding sites in the plasma membranes of rat liver appear to have the highest affinity of corticosterone, followed by cortisol and cortisone. A synthetic glucocorticoid [3H]-dexamethasone, did not show any specific binding to the liver plasma membranes. Neither dexamethasone nor nonglucocorticoids such as estradiol given simultaneously affected [3H] cortisol binding to the plasma membranes.