Insect development is controlled by the combined actions of ecdysteroid and juvenile hormones. Transcriptional control by ecdysteroid hormones is mediated via two nuclear receptor superfamily members, ecdysone receptor (EcR) and its heterodimeric partner, ultraspiracle (USP). Although the ecdysteroid hormone 20-hydroxyecdysone acts as an EcR ligand and activates transcription through EcR/USP heterodimers, the activity of juvenile hormones, such as Juvenile hormone III (JH III), and methoprenic acid (MA) via USP remains unclear. Here, we demonstrate that juvenile hormones act as USP ligands and exhibit suppressive effects on ecdysone-dependent EcR transactivation. JH III- and MA-bound USP markedly repressed ecdysone-dependent EcR transactivation through shifting of the USP ligand-binding domain alpha-helix 12 without affecting EcR/USP heterodimerization or DNA binding. Moreover, transcriptional repression by USP ligands was attenuated by a histone deacetylation inhibitor. Our results suggested that juvenile hormones serve as USP ligands that antagonize EcR-mediated ecdysone actions through the recruitment of histone deacetylase complexes. (C) 2004 Elsevier Inc. All rights reserved.