Thyroxin (T4) is essential for brain development. It is synthesized outside the central nervous system (CNS), and enters brain either directly across the blood-brain barrier or indirectly via the choroid plexus epithelium (CPE), the blood-cerebrospinal fluid (CSF) barrier. However the transport mechanism/s of T4 across the CPE remains unclear. The current study characterised the steady-state T4 transport from blood to CSF, across the CPE from the lateral ventricle of sheep. Sheep were anaesthetized (20 mg.kg-1 thiopentone, I.V.) heparinised and exsanguinated. The brain was quickly removed and the CPE perfused via the internal choroidal arteries with albumin Ringer solution containing tracer levels of 125I-T4(90 pM) and 14C-mannitol, a non-transportable vascular permeability marker. All procedures were in accordance with Animals (Scientific Procedures) Act 1986. After 1 hr of perfusion, steady state extraction of 125I-T4 was achieved, then either 10 µM verapamil (inhibitor of MDR1 (multidrug resistance 1)) or 100 µM probenecid (inhibitor of Oatp2 (organic anion polypeptide 2)) was added to the Ringer, and 125I-T4 extraction measured for a further 45 min. In the presence of verapamil the net 125I-T4 extraction was reduced by about one third (34.3 ± 2.0%, n=10) compared to controls. Probenecid had a greater effect, reducing the extraction by almost half (P<0.05, student paired t-Test). We conclude that 125I-T4 may be transported across the CPE of the sheep via Oatp2 located at the basolateral membrane and via MDR1, localized sub-apically (inner membrane of the apical surface of the CPE). However, further work is needed to determine the role of other transporters, such as Oatp1 & Oatp3, in T4 transport across the CPE of the sheep. These transporters may play a significant role in the maintenance of T4 concentration in CSF and brain interstitial fluid.