Transport of various amphipathic organic compounds is mediated by organic anion transporting polypeptides (OATPs in humans, Oatps in rodents), which belong to the solute carrier family 21A (SLC21A/Slc21a). Several of these transporters exhibit a broad and overlapping substrate specificity and are expressed in a variety of different tissues. We have isolated and functionally characterized OATP-F (SLC21A14), a novel member of the OATP family. The cDNA (3059 bp) contains an open reading frame of 2136 bp encoding a protein of 712 amino acids. Its gene containing 15 exons is located on chromosome 12p12. OATP-F exhibits 47–48% amino acid identity with OATP-A, OATP-C, and OATP8, the genes of which are clustered on chromosome 12p12. OATP-F is predominantly expressed in multiple brain regions and Leydig cells of the testis. OATP-F mediates high affinity transport of T₄ and reverse T₃ with apparent K_m values of approximately 90 nm and 128 nm, respectively. Substrates less well transported by OATP-F include T₃, bromosulfophthalein, estrone-3-sulfate, and estradiol-17β-glucuronide. Furthermore, OATP-F-mediated T₄ uptake could be cis-inhibited by l-T₄ and d-T₄, but not by 3,5-diiodothyronine, indicating that T₄ transport is not stereospecific, but that 3′,5′-iodination is important for efficient transport by OATP-F. Thus, in contrast to most other family members, OATP-F has a more selective substrate preference and may play an important role in the disposition of thyroid hormones in brain and testis.