Thyroid hormone (TH) has long been known to be essential for normal brain development in both humans and animals, and increasing evidence suggests that environmental components may disrupt TH signaling. In the present study, two-hybrid yeast bioassay and chemical analysis were used to evaluate and identify thyroid-receptor (TR) disruptors in water from the Guanting Reservoir, Beijing, China. Modified yeast bioassay showed that the water samples could affect TH signaling. The bioassay-derived amiodarone hydrochloride equivalents ranged from 33.8 ± 3.3 to 308.5 ± 31.8 µg/L. Solid-phase extraction was used to separate the organic extracts, which were subjected to bioassay and chemical analysis. The organic extracts significantly antagonized the TR, which accounted for >86.0 % of the total effects. Thus, organic extracts may play a major role in the TR-disruption activity of the water. Phenols, organochlorine pesticides, and phthalate esters were detected in the organic extracts. Chemical analysis and toxic-equivalent calculation showed that a major cause of the TR antagonism of the water was dibutyl phthalate (80.1 to 122.7 %).