The present invention relates, at least in part, to the use of substituted tetracycline compounds for regulation of expression of nucleic acids operably linked to a tetracycline operator system. The invention pertains to the compounds used in a regulatory system which utilizes components of the Tet repressor/operator/inducer system of prokaryotes to regulate gene expression in cells. Use of these tetracycline compounds, as featured in the methods of the invention, result in improved dose-response results when compared to those for e.g., tetracycline and doxycycline. Certain methods of the invention thus allow for enhanced control of the Tet repressor/operator/inducer system in regulating gene expression in cells.