Mutations of the X-linked thyroid hormone (TH) transporter (monocarboxylate transporter, MCT8) produce in humans unusual abnormalities of thyroid function characterized by high serum T₃ and low T₄ and rT₃. The mechanism of these changes remains obscure and raises questions regarding the regulation of intracellular availability and metabolism of TH. To study the pathophysiology of MCT8 deficiency, we generated Mct8 knockout mice. Male mice deficient in Mct8 (Mct8^−/y) replicate the thyroid abnormalities observed in affected men. TH deprivation and replacement with l-T₃ showed that suppression of TSH required higher serum levels T₃ in Mct8^−/y than wild-type (WT) littermates, indicating hypothalamus and/or thyrotroph resistance to T₃. Furthermore, T₄ is required to maintain the high serum T₃ level because the latter was not different between the two genotypes during administration of T₃. Mct8^−/y mice have 2.3-fold higher T₃ content in liver associated with 6.1- and 3.1-fold increase in deiodinase 1 mRNA and enzymatic activity, respectively. The relative T₃ excess in liver of Mct8^−/y mice produced a decrease in serum cholesterol (79 ± 18 vs. 137 ± 38 mg/dl in WT) and an increase in alkaline phosphatase (107 ± 23 vs. 58 ± 3 U/liter in WT) levels. In contrast, T₃ content in cerebrum was 1.8-fold lower in Mct8^−/y mice, associated with a 1.6- and 10.6-fold increase in D2 mRNA and enzymatic activity, respectively, as previously observed in TH-deprived WT mice. We conclude that cell-specific differences in intracellular TH content due to differences in contribution of the various TH transporters are responsible for the unusual clinical presentation of this defect, in contrast to TH deficiency.