Gene regulation by tetracyclines has become a widely-used tool to study gene functions in pro- and eukaryotes. This regulatory system originates from Gram-negative bacteria, in which it fine-tunes expression of a tetracycline-specific export protein mediating resistance against this antibiotic. This review attempts to describe briefly the selective pressures governing the evolution of tetracycline regulation, which have led to the unique regulatory properties underlying its success in manifold applications. After discussing the basic mechanisms we will present the large variety of designed alterations of activities which have contributed to the still growing tool-box of components available for adjusting the regulatory properties to study gene functions in different organisms or tissues. Finally, we provide an overview of the various experimental setups available for pro- and eukaryotes, and touch upon some highlights discovered by the use of tetracycline-dependent gene regulation.