The transport of [l25I]T3., and [l25I]T4 through the brain capillary wall, i.e. the blood-brain barrier, was studied in barbiturate-anesthetized rats using a tissue-sampling-carotid injection technique. The percent extraction of unidirectional influx of thyroid hormone during a single pass through the brain was measured relative to a highly diffusible [3H]water reference. The Km of T3 transport was 1.1 μM; T3 transport was inhibited by T4 (Ki = 2.6 μM), rT3;, (Ki = 5.4 μM), and D-T3 but not by 1000 μM concentrations of tyrosine, leucine, or potassium iodide. Bovine albumin also inhibited blood-brain barrier transport of T3). The fractional inhibition of T3 transport by albumin was a measure of the binding of T3 by albumin in vivo, i.e. in the presence of a competing binding system, the BBB T3 carrier. The apparent dissociation constant (Kd) of albumin binding of T3 at the brain capillary level (76 μM) was 16-fold greater than the K3 of albumin binding of T3 in vitro (4.7 μM), as determined by equilibrium dialysis. A model was derived that allowed for the in vivo application of the principles of the competitive ligand-binding assay; given apparent Kd = Kd (1 4- C/Km), the local capillary T3-binding capacity (C) may be calculated from the known values for apparent Kd) Kd, and Km. Based on the relative binding index (C/Km) of BBB binding of T3 vs. the binding index of physiological concentrations of albumin, it may be estimated that about 10% of albumin-bound T3 (which is 10-fold the fraction of dialyzable T3) is transported into the brain on a single pass in the rat.