Thyroid hormones (THs) are transported across cell membranes by different transmembrane transporter proteins. In previous studies, we showed marked 3,3'-diiodothyronine (3,3'-T2) but moderate T3 uptake by the L-type amino acid transporter 2 (Lat2). We have now studied the structure-function relationships of this transporter and TH-like molecules. Our Lat2 homology model is based on 2 crystal structures of the homologous 12-transmembrane helix transporters arginine/agmatine antiporter and amino acid/polyamine/organocation transporter. Model-driven mutagenesis of residues lining an extracellular recognition site and a TH-traversing channel identified 9 sensitive residues. Using Xenopus laevis oocytes as expression system, we found that side chain shortening (N51S, N133S, N248S, and Y130A) expanded the channel and increased 3,3'-T2 transport. Side chain enlargements (T140F, Y130R, and I137M) decreased 3,3'-T2 uptake, indicating channel obstructions. The opposite results with mutations maintaining (F242W) or impairing (F242V) uptake suggest that F242 may have a gating function. Competitive inhibition studies of 14 TH-like compounds revealed that recognition by Lat2 requires amino and carboxylic acid groups. The size of the adjacent hydrophobic group is restricted. Bulky substituents in positions 3 and 5 of the tyrosine ring are allowed. The phenolic ring may be enlarged, provided that the whole molecule is flexible enough to fit into the distinctly shaped TH-traversing channel of Lat2. Taken together, the next Lat2 features were identified 1) TH recognition site; 2) TH-traversing channel in the center of Lat2; and 3) switch site that potentially facilitates intracellular substrate release. Together with identified substrate features, these data help to elucidate the molecular mechanisms and role of Lat2 in T2 transport.