Precise control of transgene expression would markedly facilitate certain applications of gene therapy. To regulate expression of a transferred gene in response to an exogenous compound in vivo, we modified the ecdysone-responsive system. We combined the advantages of the Drosophila (DmEcR) and the Bombyx ecdysone receptor (BmEcR) by creating a chimeric Drosophila/Bombyx ecdysone receptor (DB–EcR) that preserved the ability to bind to the modified ecdysone promoter without exogenous retinoid X receptor (RXR). In cultured cells, DB–EcR effectively mediates ligand-dependent transactivation of a reporter gene at lower concentrations of the chemical ecdysone agonist GS-E than VgRXR (DmEcR + RXR). Transgene delivery in vivo was achieved by intramyocardial injection of recombinant adenovirus vectors in adult rats. Upon stimulation with GS-E, DB–EcR potently (>40-fold induction) activated gene expression in vivo while VgRXR was not induced. This hybrid ecdysone receptor represents an important new tool for in vivo transgene regulation with potentially diverse applications in somatic and germline transfer.