The present study was aimed at investigating the involvement of mouse organic anion transporting polypeptide 14 (mOatp14) in the uptake of T₄ across the blood-brain barrier. Functional expression of mOatp14 in HEK293 cells revealed that T₄ and rT₃ are high affinity substrates of mOatp14 (Michaelis constant, 0.34 and 0.46 μm, respectively), and the specific uptake of T₃ was 4-fold less than that of T₄ and rT₃. Taurocholate, probenecid, and estrone-3-sulfate were moderate inhibitors for mOatp14, whereas digoxin (substrate of Oatp2), benzylpenicillin (substrate of Oat3), and large neutral amino acids had no effect. mOatp14 is widely expressed throughout the brain, except for the cerebellum. The expression of mOatp14 in the isolated brain capillaries and the choroid plexus was shown by Western blot. The uptake clearance of T₄ by the cerebral cortex determined using the in situ brain perfusion technique in mice was 580 μl/min·g tissue, 3-fold greater than that by the cerebellum, and a saturable component (Michaelis constant, 1.0 μm) accounts for the major fraction of the total uptake. Taurocholate inhibited the uptake of T₄ by the cerebral cortex completely, but the inhibition by estrone-3-sulfate was partial (50%). These results suggest that transporters play a predominant role in the delivery of T₄ to the brain, and mOatp14 accounts for estrone-3-sulfate inhibitable fraction, at least partly. The absence of inhibition by digoxin, benzylpenicillin, leucine, and 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid for the uptake of T₄ by the cerebral cortex suggests the presence of other unknown transporter for T₄ uptake by the brain. Immunohistochemical staining revealed basolateral localization of mOatp14 in the choroid plexus in which it may also play a role in T₄ uptake.