Transport of thyroid hormone across the cell membrane is required for thyroid hormone action and metabolism. We have investigated the possible transport of iodothyronines by the human system L amino acid transporter, a protein consisting of the human 4F2 heavy chain and the human LAT1 light chain. Xenopus oocytes were injected with the cRNAs coding for human 4F2 heavy chain and/or human LAT1 light chain, and after 2 d were incubated at 25 C with 0.01-10 microM [(125)I]T(4), [(125)I]T(3), [(125)I]rT(3), or [(125)I]3,3'-diiodothyronine or with 10-100 microM [(3)H]arginine, [(3)H]leucine, [(3)H]phenylalanine, [(3)H]tyrosine, or [(3)H]tryptophan. Injection of human 4F2 heavy chain cRNA alone stimulated the uptake of leucine and arginine due to dimerization of human 4F2 heavy chain with an endogenous Xenopus light chain, but did not affect the uptake of other ligands. Injection of human LAT1 light chain cRNA alone did not stimulate the uptake of any ligand. Coinjection of cRNAs for human 4F2 heavy chain and human LAT1 light chain stimulated the uptake of phenylalanine > tyrosine > leucine > tryptophan (100 microM) and of 3,3'-diiodothyronine > rT(3) approximately T(3) > T(4) (10 nM), which in all cases was Na(+) independent. Saturation analysis provided apparent Michaelis constant (K(m)) values of 7.9 microM for T(4), 0.8 microM for T(3), 12.5 microM for rT(3), 7.9 microM for 3,3'-diiodothyronine, 46 microM for leucine, and 19 microM for tryptophan. Uptake of leucine, tyrosine, and tryptophan (10 microM) was inhibited by the different iodothyronines (10 microM), in particular T(3). Vice versa, uptake of 0.1 microM T(3) was almost completely blocked by coincubation with 100 microM leucine, tryptophan, tyrosine, or phenylalanine. Our results demonstrate stereospecific Na(+)-independent transport of iodothyronines by the human heterodimeric system L amino acid transporter.