Background Monocarboxylate transporter 8 (MCT8) is the most specific thyroid hormone transporter identified to date, deficiency of which has been associated with severe intellectual and motor disability and abnormal serum thyroid function tests. However, it is currently unknown if MCT8, like other thyroid hormone transporters, also accepts additional substrates, and if disruption of their transport may contribute to the observed phenotype. Methods In this study, we aimed to identify such substrates by applying LC-MS-based metabolome analysis in lysates of control and MCT8-overexpressing Xenopus oocytes. A subset of identified candidate substrates was validated by direct transport studies in transiently transfected COS-1 cells and human fibroblasts which endogenously express MCT8. Moreover, transport characteristics were determined, including transport saturation and cis-inhibition potency of thyroid hormone transport. Results Metabolome analysis identified 21 m/z ratios, corresponding to 87 candidate metabolites, with a 2.0-times differential abundance in MCT8-injected oocytes compared to controls. These metabolites included 3,5-diiodotyrosine (DIT) and several amino acids, including glutamate and glutamine. In accordance, MCT8-expressing COS-1 cells had 2.2-times lower intracellular accumulation of [125I]-DIT compared to control cells. This effect was largely blocked in the presence of T3 (IC50: 2.5±1.5 µM) or T4 (IC50: 5.8±1.3 µM). Conversely, increasing concentrations of DIT enhanced the accumulation of T3 and T4. The MCT8-specific inhibitor silychristin increased the intracellular accumulation of DIT in human fibroblasts. COS-1 cells expressing MCT8 also exhibited a 50%-reduction in intracellular accumulation of [125I]-3-monoiodotyrosine (MIT). In contrast, COS-1 cells expressing MCT8 did not alter the intracellular accumulation of [3H]-glutamate or [3H]-glutamine in. However, studies in human fibroblasts showed a 1.5-1.9-times higher glutamate uptake in control fibroblasts compared to fibroblasts derived from patients with MCT8 deficiency, which was not affected in the presence of silychristin. Conclusions Taken together, our results suggest that the iodotyrosines DIT and MIT can be exported by MCT8. MIT and DIT interfere with MCT8-mediated transport of thyroid hormone in vitro, and vice versa. Future studies should elucidate if MCT8, being highly expressed in thyroidal follicular cells, also transports iodotyrosines in vivo.