Cellular entry of thyroid hormone is mediated by plasma membrane transporters. We have identified rat monocarboxylate transporter 8 (MCT8) as an active and specific thyroid hormone transporter. The MCT8 gene is located on the X-chromosome. The physiological relevance of MCT8 has been demonstrated by the identification of hemizygous mutations in this gene in males with severe psychomotor retardation and elevated serum T₃ levels. We have characterized human (h) MCT8 by analysis of iodothyronine uptake and metabolism in cell lines transiently transfected with hMCT8 cDNA alone or together with cDNA coding for iodothyronine deiodinase D1, D2, or D3. MCT8 mRNA was detected by RT-PCR in a number of human cell lines as well as in COS1 cells but was low to undetectable in other cell lines, including JEG3 cells. MCT8 protein was not detected in nontransfected cell lines tested by immunoblotting using a polyclonal C-terminal hMCT8 antibody but was detectable in transfected cells at the expected size (61 kDa). Transfection of COS1 and JEG3 cells with hMCT8 cDNA resulted in 2- to 3-fold increases in uptake of T₃ and T₄ but little or no increase in rT₃ or 3,3′-diiodothyronine (3,3′-T₂) uptake. MCT8 expression produced large increases in T₄ metabolism by cotransfected D2 or D3, T₃ metabolism by D3, rT₃ metabolism by D1 or D2, and 3,3′-T₂ metabolism by D3. Affinity labeling of hMCT8 protein was observed after incubation of intact transfected cells with N-bromoacetyl-[¹²⁵I]T₃. hMCT8 also facilitated affinity labeling of cotransfected D1 by bromoacetyl-T₃. Our findings indicate that hMCT8 mediates plasma membrane transport of iodothyronines, thus increasing their intracellular availability.