The uptake of corticosterone by highly purified plasma membrane vesicles of rat liver was studied by a rapid-centrifugation technique which allows uptake measurements within 5 s. The vesicles are free of soluble cytoplasmic constituents. Therefore, association of hormone with the vesicle is attributed entirely to components of the vesicle-membrane. Half maximal uptake is reached at 8 s at 21 degrees C. At 15 degrees C transition of the lipid state in the membrane leads to a decrease of uptake, a characteristic property common to membrane mediated processes. The uptake of corticosterone is saturable and reversible but does not follow normal saturation kinetics. The apparent dissociation constants of three uptake systems bear direct relation to the concentration of free corticosterone in rat plasma (4-16 nM) supporting a physiological role for the system. Uptake of corticosterone decreases with decreases in vesicular volume; about 50% of the hormone is bound specifically and 50% is transported to the lumen of the vesicle. Since outflow of intravesicular hormone also occurs readily, the uptake and transport is proposed to be mediated by putative "carriers". The "carrier" preferentially transports glucocorticoids; dexamethasone is not taken up by this putative molecule. Steroids with 5 alpha conformation are more potent inhibitors of the "carrier" for corticosterone than 5 beta-steroids. Androgens and estrogens are weak competitors of corticosterone. The affinity of the "carrier" for several hormones differs considerably from that of the cytoplasmic receptor. Morris hepatoma cells (MH 3924) do not take up corticosterone. Our results prompt us to propose the hypothesis that the transport function of the "carrier" and the binding of the hormone by the cytoplasmic receptor are two different entities; perturbation of the "carrier" may lead to steroid unresponsiveness. Normal expression of steroid hormone activity is manifested in the concerted action of the functionally sound cell membrane "carrier" and the intracellular receptor.