The ability to control expression of a target gene in eukaryotic cells has important applications in transgenic studies, metabolic engineering, and gene therapy. We report the development of two new gene switches in yeast, where gene expression is regulated in a ligand dependent manner. Sites within the estrogen receptor alpha ligand binding domain were targeted by sequential saturation mutagenesis to exchange specificity from 17 β-estradiol (E2, the native ligand) to two new ligands: 4,4'-dihydroxybenzil (DHB) and 2,4-di(4-hydroxyphenyl)-5-ethylthiazole (L9). Random mutagenesis was employed once the targeted strategy ceased yielding improvements. After seven rounds of mutagenesis, the ligand binding domains were seen to be highly specific for the desired ligand. Variant 7S showed a subnanomolar response to DHB, with sensitivity to E2 and L9 reduced to micromolar levels. Variant L7E showed a nanomolar response to L9, with no visible response to E2 or L9 Chimeric proteins consisting of Gal4 DNA binding domain and the engineered ligand binding domains showed an orthogonal response to 17 β-estradiol, DHB, and L9 in a transactivation assay. The independent response of a ligand binding domain to its ligand was tested by constructing gene circuits including AND and OR gates, which were monitored by the expression of green fluorescent protein. Various repression domains were evaluated for their ability to downregulate gene expression in a ligand dependent manner by creating proteins that contained a DNA binding domain, the engineered ligand responsive domain, and the repressor domain.