Stringent control of gene expression in human gene therapy strategies is important for both therapeutic and safety reasons. Replication-defective vectors derived from adenoviruses have been shown to be capable of highly efficient in vivo gene delivery to a wide variety of dividing and nondividing human cells. Here, we review the progress in the development of regulatable adenovirus vectors that allow gene expression to be tightly controlled by low concentrations of tetracyclines. As an example of the potential clinical utility of this technology, we highlight our results obtained in an immunotherapy model for prostate cancer with a tetracycline-regulatable adenovirus vector expressing the cytokine interleukin-12. Recombinant adenovirus vectors with tetracycline-regulatable gene expression provide new opportunities and improved safety for gene therapy applications in humans.