The development of nuclear hormone receptor antagonists that directly inhibit the association of the receptor with its essential coactivators would allow useful manipulation of nuclear hormone receptor signalling. We previously identified 3-(dibutylamino)-1-(4-hexylphenyl)-propan-1-one (DHPPA), an aromatic -amino ketone that inhibits coactivator recruitment to thyroid hormone receptor (TR), in a high-throughput screen. Initial evidence suggested that the aromatic -enone 1-(4-hexylphenyl)-prop-2-en-1-one (HPPE), which alkylates a specific cysteine residue on the TR surface, is liberated from DHPPA. Nevertheless, aspects of the mechanism and specificity of action of DHPPA remained unclear. Here, we report an X-ray structure of TR with the inhibitor HPPE at 2.3-Å resolution. Unreacted HPPE is located at the interface that normally mediates binding between TR and its coactivator. Several lines of evidence, including experiments with TR mutants and mass spectroscopic analysis, showed that HPPE specifically alkylates cysteine residue 298 of TR, which is located near the activation function-2 pocket. We propose that this covalent adduct formation proceeds through a two-step mechanism: first, -elimination to form HPPE; second, a covalent bond slowly forms between HPPE and TR. DHPPA represents a novel class of potent TR antagonist, and its crystal structure suggests new ways to design antagonists that target the assembly of nuclear hormone receptor gene regulatory complexes and block transcription.