The Regulated Mammalian Expression System features low basal levels, robust and rapid induction, and downregulation of gene expression in mammalian cells. The Regulated Mammalian Expression System is based on a novel on/off switch that relies on the rapid and sensitive modulation by coumerin-related compounds of a chimeric transactivator protein. Nanomolar concentrations of the antibiotic coumermycin promote homodimerization of a chimeric transactivator that, in turn, binds to lambda operator sequences located upstream of a minimal promoter driving transcription of coding sequences for a protein of interest. The levels of protein expression can be regulated by adjusting the coumermycin concentration. More significantly, this expression can be promptly and effectively switched off by adding novobiocin, which acts as an antagonist by dissociating the dimerized transactivator protein. The protein coding region of interest is cloned into either the pF12A RM Flexi Vector or pF12K RM Flexi Vector, both of which are specially designed for Regulated Mammalian (RM) protein expression. These vectors incorporate regulatory promoter sequences upstream of the protein-coding region and are compatible with the Flexi Vector System. In transient transfection paradigms, the pF12A or pF12K RM Flexi Vector containing the protein-coding region of interest is co-transfected into mammalian cells together with the pReg neo Vector. The pReg neo Vector is designed to express a chimeric transactivator protein that interacts with the regulatory promoter region in the pF12A and pF12K RM Flexi Vectors in a regulated fashion in response to coumermycin and novobiocin. Additionally, the pReg neo Vector encodes a neomycin phosphotransferase gene that allows stable cell selection and generation with the antibiotic G-418.