Phloretin, a 7,8-dihydrochalcone of plant origin, and the high molecular weight (<15,000) polyphloretinphosphate (PPP) polymers are potent inhibitors of iodothyronine monodeiodinase activity from rat liver microsomal preparations, whereas phlorizin, the 2′- O-glucoside of phloretin, is inactive. The polymers, differing in degree of phosphorylation-dependent polymerization, exhibited a concentration-dependent, and ultimately complete, inhibition of deiodinase activity with an IC 50 between 0.2 and 0.5 μg PPP/ml. Phloretin inhibition, on the other hand, was cofactor (DTE) competetive, with a K i = 0.75 μM. 2′,4′,6′,3,4-Pentahydroxychalcone, which has a substitution pattern in the A-ring identical to that of phloretin, was the only active inhibitor ( IC 50 = 8 μ M ) among several derivatives tested. The phloretin biodegradation products, phloretic acid and phloroglucinol, and its biosynthetic precursors, monomeric cinnamic acid and cinnamic acid derivatives, were inactive in concentrations up to 100 μM. The X-ray crystal structure analysis of phlorizin dihydrate showed that the molecule is planar and fully extended, similar to the conformation observed in chalcone structures that are characterized by an α, β-unsaturated bond between phenol rings. Comparison of the planar phlorizin crystal structure with a skewed or antiskewed thyroid hormone conformation revealed that the β- D-glucose moiety does not share any of the thyroid hormone's conformational space, and that the best structural homology is found with the antiskewed conformation of 3′,5′,3-triiodothyronine, the natural deiodinase substrate that also inhibits further deiodination.