The regulated expression of therapeutic genes may become crucial in gene therapy when their constitutive expression interferes with cell fate in vivo. The efficient regulation of transgene expression requires tightly controlled inducible promoters, as shown for the tetracycline regulatory system (tet-system). However, its application requires the introduction of two components into the target cell genome: the tet-responsive transactivator and the regulated expression cassette. In order to facilitate the usage of the tet-system for approaches in gene therapy, both components have to be transferred by a single vector, thus eliminating the preselection of transactivator positive cells. Published "all-in-one" vectors for regulated transgene expression display a relatively low signal-to-noise ratio, resulting in regulatory windows of around 500-fold even in selected clones. In this study, we show that a modified vector architecture combined with the introduction of new tet-responsive promoters, Ptet, improved the dynamic range of such all-in-one vectors to levels up to 14,000-fold for viral and 25,000-fold for nonviral transfer vectors in nonclonal human cell lines, and up to 2,800-fold in murine hematopoietic cell lines. This improved regulation was the result of a strong reduction of background expression in the off-state, even if cells were transduced at high multiplicity of infection, while induction remained at high levels. In addition, the results indicated that successful regulation of gene expression in different target cells depended on vector architecture as well as the choice of the Ptet-promoter.