The unidirectional influx of 3H-gonadal (progesterone, dihydrotestosterone, testosterone, estradiol) and adrenal (aldosterone, cortisol) steroid hormones into liver was studied with a tissue-sampling single-injection technique in barbiturate-anesthetized rats. Liver uptake of the steroid hormone was measured relative to [14C]butanol, a highly diffusible reference, after a single pass through the liver. Portal flow (1.4 ml.min-1.g-1) under the experimental conditions was measured with 3H2O. The extraction of unidirectional influx of all six steroid hormones was 70-100% after a bolus portal injection of labeled steroid in Ringer solution (0.1% albumin). Steroid transport was nonsaturable because 35 muM concentrations of unlabeled hormone resulted in no inhibition of liver transport. The plasma proteins (albumin and specific globulins) in serum from human (male, female, pregnancy), rat (male), and guinea pig (pregnancy) sources inhibited the liver clearance of the respective steroid hormones to a variable extent. In all cases albumin-bound steroid hormone was freely cleared by liver and, in the case of cortisol or estradiol, the fraction bound to a specific globulin was also transported into liver.