To investigate the mechanism by which thyroxine (T4) in plasma enters hepatic cells, we measured the rate constants for uptake of free T4 by the perfused rat liver and for dissociation of T4 from its plasma binding proteins. Quantitative autoradiography of liver lobules after perfusion with [125I]T4 indicated an apparent rat constant for removal of free T4 from the sinusoids of at least 1.1 +/- 0.2 s-1. Single-pass extraction of T4 from human serum was 10.6 +/- 1.7% at physiological flow rates (1 ml.min-1.g liver-1). Rate constants for dissociation of T4 from plasma binding proteins at 37 degrees C (determined by rapid filtration) were 0.017 +/- 0.002 s-1 for human thyroid hormone-binding globulin, 0.080 +/- 0.015 s-1 for human thyroid hormone-binding prealbumin, and greater than 0.5 s-1 for human albumin. To investigate the factors that determine the concentration of T4 within hepatic cells, we analyzed the above data together with data reported in the literature on the equilibrium-binding constants and the rate constant for cellular metabolism of T4. Analysis of all of these data using a previously published mathematical model leads to the following conclusions for the physiological state: 1) metabolism, not uptake, is rate limiting to removal of T4 from plasma by the liver; 2) binding equilibrium is present in the intrahepatic plasma; 3) intracellular T4 is in equilibrium with the free T4 pool in plasma (and maintenance of this equilibrium may be an important function of plasma thyroid hormone-binding proteins); and 4) the concentration of T4 within the liver is proportional to the concentration of free T4 in the plasma. Our data do not allow us to determine definitively whether hepatic uptake of T4 occurs only from the free T4 pool in plasma or also from the protein-bound pool by interaction of one or more of the binding proteins with the liver cell. However, mathematical analysis indicates that this distinction is irrelevant to steady-state intracellular hormone concentrations when equilibrium exists between the plasma and cytosolic pools of hormone.