The tetracycline-responsive expression system is based on the ability of the chimeric tTA and rtTA transactivators to stimulate specifically transcription from a companion synthetic CMV* or TK* promoter element, and can provide tightly regulated gene expression that can be induced up to five orders of magnitude in cultured cells and transgenic mice. A major problem with the system is that high level expression of the tTA or rtTA transactivators causes cellular toxicity. Under conditions of prolonged expression this results in selective pressure against the stable incorporation of vectors expressing the tTA or rtTA transactivators, and makes the generation of stable cell lines and transgenic mice problematic. In this report we describe the development of a set of autoregulated bi-directional expression vectors in which the weaker TK* promoter is used to direct expression of the rtTA or tTA transactivator and the stronger CMV* element is used to direct cDNA expression. In this format the transactivator and response elements are encoded on the same vector, which simplifies the system and ensures that gene expression is effectively skewed in favor of the cDNA while maintaining a continuously low level of transactivator expression. We find that such an autoregulated system works equally well for both the tTA and rtTA transactivators, provided that they contain a nuclear localization signal. Similar to other versions of the tetracycline-responsive expression system, gene expression is tightly regulated and can be efficiently switched between the off and on expression states by doxycycline. In contrast with other tetracycline-responsive systems, however, expression of the rtTA and tTA transactivators from the autoregulated TK* promoter is low enough such that there is no cellular toxicity associated with either expression state. By incorporating a selectable marker into these vectors, all of the components required for using the system are now contained on a single plasmid construct, and we find that this format provides a more reliable and greatly simplified method for the generation of stable cell lines.