The postulate that thyroxine (T4) in plasma enters tissues by protein-mediated transport or enhanced dissociation from plasma-binding proteins leads to the conclusion that almost all T4 uptake by tissues in the rat occurs via the pool of albumin-bound T4 (Pardridge, W. M., B. N. Premachandra, and G. Fierer. 1985. Am. J. Physiol. 248:G545-G550). To directly test this postulate, and to test more generally whether albumin might play a special role in T4 transport in the rat, we performed in vivo kinetics studies in six Nagase analbuminemic rats and in six control rats, all of whom had similar serum T4 concentrations and percent free T4 values. Evaluation of the plasma disappearance curves of simultaneously injected 125I-T4 and 131I-albumin indicated that the flux of T4 from the extracellular compartment into the rapidly exchangeable intracellular compartment was similar in the analbuminemic rats (51 +/- 21 ng/min, mean +/- SD) and in the control rats (54 +/- 15 ng/min), as was the size of the rapidly exchangeable intracellular pool of T4 (1.13 +/- 0.53 vs. 1.22 +/- 0.36 micrograms). This latter finding was confirmed by direct analysis of tissue samples (liver, kidney, and brain). We also performed in vitro kinetics studies using the isolated perfused rat liver. The single-pass fractional extraction by normal rat liver of T4 in pooled analbuminemic rat serum was indistinguishable from that of T4 in pooled control rat serum (10.9 +/- 3.3%, n = 3, vs. 11.4 +/- 3.4%). When greater than 98% of the albumin was removed from normal rat serum by chromatography with Affi-Gel blue, the single-pass fractional extraction of T4 (measured by a bolus injection method) did not change (16.3 +/- 2.1%, n = 5, vs. 15.2 +/- 2.5%). These data provide the first valid experimental test of the enhanced dissociation hypothesis and indicate that there is no special, substantive role for albumin in T4 transport in the rat.