Technical Abstract: Chemical inducible gene regulation systems provide essential tools for the precise regulation of transgene expression in plants and animals. We have recent developed a two-hybrid ecdysone receptor (EcR) gene regulation system that works in conjunction with the retinoid X receptor of Locusta migratoria RXR (LmRXR). To further improve some of the observed drawbacks in the original two-hybrid and monopartite gene switches, we tested several EcR mutants that were generated by changing single or double amino acid residues in the ligand binding domain of Choristoneura fumiferana EcR (CfEcR). Based on the transient expression data obtained on the functioning of different EcR mutants, we selected a double mutant, where the amino acids were changed from isoleucine to valine and glutamate to tyrosine at positions 395 and 415 (called CfEcRVY mutant), respectively and tested these further in stable transformation experiments. In two-hybrid experiments the CfEcRVY mutant did not improve expression over the wild-type CfEcR ligand binding domain. In the monopartite gene switch experiments, the VGCfEVY mutant the sensitivity of the mutant switch was improved by 125 to 15625 times compared to the wild-type. The utility of VGCfEVY switch was further shown in regulating expression of Arabidopsis zinc finger protein 11 (AtZFP11) gene in both tobacco and Arabidopsis transgenic plants.