Molecular switch systems that activate gene expression by a small molecule are effective technologies that are widely used in applied biological research. Previously, two orthogonal ligand receptor pairs (OLRP) were developed as potential molecular switch systems by modifying nuclear receptors, ligand-activated transcription factors, to bind and activate gene expression with the synthetic ligand LG335 and not with the natural ligand 9-cis retinoic acid (9cRA). The two OLRP previously discovered were RXR variant 130 (I268A, I310A, F313A, and L436F) (also known as GR130) and the RXR variant QCIMFI (Q275C, I310M, and F313I) and (also known as GRQCIMFI). The OLRP were further developed into molecular switches to provide controlled gene expression and potentially benefit gene therapy applications by replacing the DNA binding domain (DBD) with a Gal4 DBD, a yeast transcription factor. Both molecular switches are able to bind Gal4 RE in response to LG335 and activate expression of a luciferase or GFP reporter gene in either a two- or one-component system. When characterizing the GR130 variant in the two-component system, no activation was observed with the natural ligand 9cRA, and the variant displayed a 19±5-fold activation and a 50 nM EC50 value in the presence of LG335. When the GRQCIMFI variant was evaluated in the two-component system, activation was observed in the presence of LG335 with a 10 nM EC50 value and a 6±2-fold induction, and 9cRA induced activation only at the highest concentration. The GRQCIMFI variant was also characterized with the one-component system containing the reporter gene GFP in a transient transfection as well as through retroviral transduction, displaying green fluorescence in 30% of the cells in the presence of 10 µM LG335. Several attempts were made to improve the molecular switch system. The VP16 activation domain was fused to GRQCIMFI in an effort to increase the fold induction; however, the addition of the VP16 created a constitutively active protein. Another approach to improve the molecular switch incorporated error-prone PCR to discover a new variant, Q275C, I310M, F313I, L455M (QCIMFILM), which displayed a 10-fold increase in sensitivity towards LG335 with a 5 nM EC50 value. Examination of the L455 position in the crystal structure of RXR revealed this residue is located outside of the ligand binding pocket on helix 12 (H12), but is able to significantly enhance receptor function. In fact, the single variant, L455M, was able to enhance receptor activation, compensate for a nonfunctional variant, as well as influence coactivator association. The long-term goal of this research is to develop a gene regulation system that would be used in human gene therapy trials. In the process of creating this system a deeper assessment of the nuclear receptor structure and function is made, which can be used for the enhancement and development of transcriptional regulation mechanisms.