Previously, we developed single adenovirus (Ad) vectors that contained the gene of interest in the E1 deletion region and the transactivator gene for the tetracycline-controllable expression system in the E3 deletion region. In the present study, we improved the Ad vector-mediated tetracycline-controllable expression system by the fiber modification of Ad. We developed fiber-modified Ad vectors containing the tet-off system, which are effective in overcoming the limitations of conventional Ad vectors, specifically their inefficient gene transfer into cells lacking the primary receptor, the coxsackievirus and adenovirus receptor (CAR). Ad vectors containing the tet-off system with an Arg-Gly-Asp (RGD) peptide in the HI loop of the fiber knob or the Ad type 35 fiber greatly improved transduction efficiency (more than 1-2-log orders) into the cells lacking CAR expression but expressing alphav integrin or CD46, respectively. They exhibited vastly higher regulation of gene expression by doxycycline. The combination of fiber-modified Ad vectors and the tetracycline-controllable expression system should offer a powerful tool for gene therapy and gene transfer experiment.