Control of gene expression in eukaryotic cells is clearly important in many applications including modifications of the level of a therapeutic gene product. For effective gene delivery and regulation, the regulatory system must be contained on a single vector and it must exhibit high transgene expression on induction and low basal expression on repression. Here, we have investigated several self-contained vectors carrying both the tetracycline-controlled transactivator (tTA) and a potentially therapeutic gene in transient studies. An enhancerless positive feedback regulatory vector (pSiaIV) transcribing both tTA and mGM-CSF from a modified tTA-responsive bidirectional promoter demonstrated over 200-fold gene regulation in HeLa cells. This was comparable to the degree of regulation obtained on cotransfection of vectors expressing tTA and tTA-responsive mGM-CSF. The maximal transcriptional activity of pSiaIV was comparable to that of CMV IE promoter and its basal activity as low as the leakiness of the tetracycline-responsive promoter (tRP) in several commonly used cell lines, resulting in 47- to 328-fold regulation. Furthermore, pSiaIV also showed efficient regulation in stable cells. Overall, the positive feedback regulatory system (PFRS) offers efficient gene regulation which is suitable for most applications, especially gene therapy.