In patients, inactivating mutations in the gene encoding the thyroid hormone-transporting monocarboxylate transporter 8 (Mct8) are associated with severe mental and neurological deficits and disturbed thyroid hormone levels. The latter phenotype characterized by high T₃ and low T₄ serum concentrations is replicated in Mct8 knockout (ko) mice, indicating that MCT8 deficiency interferes with thyroid hormone production and/or metabolism. Our studies of Mct8 ko mice indeed revealed increased thyroidal T₃ and T₄ concentrations without overt signs of a hyperactive thyroid gland. However, upon TSH stimulation Mct8 ko mice showed decreased T₄ and increased T₃ secretion compared with wild-type littermates. Moreover, similar changes in the thyroid hormone secretion pattern were observed in Mct8/Trhr1 double-ko mice, which are characterized by normal serum T₃ levels and normal hepatic and renal D1 expression in the presence of very low T₄ serum concentrations. These data strongly indicate that absence of Mct8 in the thyroid gland affects thyroid hormone efflux by shifting the ratio of the secreted hormones toward T₃. To test this hypothesis, we generated Mct8/Pax8 double-mutant mice, which in addition to Mct8 lack a functional thyroid gland and are therefore completely athyroid. Following the injection of these animals with either T₄ or T₃, serum analysis revealed T₃ concentrations similar to those observed in Pax8 ko mice under thyroid hormone replacement, indicating that indeed increased thyroidal T₃ secretion in Mct8 ko mice represents an important pathogenic mechanism leading to the high serum T₃ levels.