Specific gene inhibition in mammalian cells can be achieved by the use of small interfering RNA molecules (siRNA). These siRNA molecules can be chemically synthesized and transfected into cells, or directly expressed intracellularly from a plasmid DNA by the function of the cellular RNA polymerase III. We report here that the latter concept can be incorporated into an adenovirus vector to achieve specific gene inhibition in mammalian cells. As an examination of this approach, we have prepared an adenoviral vector capable of expressing siRNA molecules targeting p53 or VprBP/KIAA0800, a cellular protein that interacts with the HIV auxiliary protein Vpr. In both cases, specific reduction in the target protein level was observed after adenoviral infection. The reduction in the protein level was correlated with a specific reduction in the mRNA level. Since many cell types can be efficiently infected by adenoviruses, adenoviral vectors may serve as a useful alternative to other methods for siRNA delivery and gene inhibition, especially when the target cells are refractory to transfection by DNA or RNA.