The ability to control (trans)gene expression is important both for basic biological research and applications such as gene therapy. In vivo use of the inducible tetracycline (Tc)-regulated gene expression system (Tet-On system) is limited by its low sensitivity for the effector doxycycline (dox). We used viral evolution to optimize this Escherichia coli-derived regulatory system for its function in mammalian cells. The components of the Tet-On system (the transcriptional activator rtTA and its tetO DNA binding site) were incorporated into the human immunodeficiency virus (HIV)-1 genome to control viral replication. Prolonged culturing of this HIV-rtTA virus resulted in virus variants that acquired mutations in the rtTA gene. Some of these mutations enhance the transcriptional activity and dox-sensitivity of the rtTA protein. This improvement was observed with different tetO-containing promoters and was independent of the episomal or chromosomal status of the target gene. Combination of these beneficial mutations resulted in greatly improved rtTA variants that are seven-fold more active and 100-fold more dox-sensitive than the original Tet-On system. Furthermore, some of the new Tet-On systems are responsive to Tc and minocycline. Importantly, these rtTA variants show no activity in the absence of dox. The optimized rtTA variants are particularly useful for in vivo applications that require a more sensitive or more active Tet-On system.