Endocrine disrupting chemicals (EDC), either plant constituents or contaminants deriving from industrial products, may interfere with the thyroid hormone axis. Here, we examined if selected EDC inhibit the key reactions of thyroid hormone biosynthesis catalyzed by thyroperoxidase (TPO). We used a novel in vitro assay based on human recombinant TPO (hrTPO) stably transfected into the human thyroid carcinoma cell line FTC-238. F21388 (synthetic flavonoid), bisphenol A (building block for polycarbonates) and the UV filter benzophenone 2 (BP2) inhibited hrTPO. BP2 is contained in numerous cosmetics of daily use and may be in regular contact with human skin. Half maximal inhibition in the guaiacol assay occurred at 450 nmol/L BP2, a concentration 20 and 200-fold lower than those required in case of the TPO-inhibiting anti-thyroid drugs methimazole and propylthiouracil, respectively. 300 nmol/L BP2 combined with the thyroperoxidase substrate H₂O₂ (10 µmol/L) inactivated hrTPO; this was, however, prevented by µmolar amounts of iodide. BP2 did not inhibit iodide uptake into FRTL-5 cells. In BP2-treated rats (333 and 1000 mg/kg body weight), serum total T4 was significantly decreased and serum thyrotropin was significantly increased. TPO activities in the thyroids of treated animals were unchanged, a finding also described for methimazole and propylthiouracil. Thus, EDC, most potently BP2, may disturb thyroid hormone homeostasis by inhibiting or inactivating TPO, effects that are even more pronounced in the absence of iodide. This new challenge for endocrine regulation must be considered in the context of a still prevailing iodide deficiency in many parts of the world.