The thyroid hormone-disrupting activity of hydroxylated PCBs was examined. 4-Hydroxy-2,2′,3,4′,5,5′-hexachlorobiphenyl (4-OH-2,2′,3,4′,5,5′-HxCB), 4-hydroxy-3,3′,4′,5-tetrachlorobiphenyl (4-OH-3,3′,4′,5-TCB) and 4,4′-dihydroxy-3,3′,5,5′-tetrachlorobiphenyl (4,4′-diOH-3,3′,5,5′-TCB), which have been detected as metabolites of PCBs in animals and humans, and six other 4-hydroxylated PCBs markedly inhibited the binding of triiodothyronine (1 × 10 −10 M) to thyroid hormone receptor (TR) in the concentration range of 1 × 10 −6 to 1 × 10 −4 M. However, 4-hydroxy-2′,4′,6′-trichlorobiphenyl (4-OH-2′,4′,6′-TCB), 3-hydroxy-2,2′,5,5′-tetrachlorobiphenyl, 4-hydroxy-2,2′,5,5′-tetrachlorobiphenyl, 4-hydroxy-2,3,3′,4′-tetrachlorobiphenyl, 2,3′,5,5′-tetrachlorobiphenyl and 2,3′,4′,5,5′-pentachlorodiphenyl did not show affinity for TR. The thyroid hormonal activity of PCBs was also examined using rat pituitary cell line GH3 cells, which grow and release growth hormone in a thyroid hormone-dependent manner. 4-OH-2,2′,3,4′,5,5′-HxCB, 4,4′-diOH-3,3′,5,5′-TCB and 4-OH-3,3′,4′,5-TCB enhanced the proliferation of GH3 cells and stimulated their production of growth hormone in the concentration range of 1 × 10 −7 to 1 × 10 −4 M, while PCBs which had no affinity for thyroid hormone receptor were inactive. In contrast, only 4-OH-2′,4′,6′-TCB exhibited a significant estrogenic activity using estrogen-responsive reporter assay in MCF-7 cells. However, the 3,5-dichloro substitution of 4-hydroxylated PCBs markedly decreased the estrogenic activity. These results suggest that, at least for the 17 PCB congeners and hydroxylated metabolites tested, a 4-hydroxyl group in PCBs is essential for thyroid hormonal and estrogenic activities, and that 3,5-dichloro substitution favors thyroid hormonal activity, but not estrogenic activity.