Recombinant adenoviruses have been extensively applied in basic research and gene therapy. Recently, there is an increase in neurobiological studies using recombinant adenovirus technology to manipulate gene expression in neurological systems. Recombinant adenovirus technology provides a useful tool for investigating the function of a gene of interest, neurocircuitries and the delivery of genes for therapy. In order to take full advantage of this technique, it is important to understand the strategies for the design and application of the virus. The goal of this chapter is to provide a practical protocol on the application of recombinant adenovirus for neurobiological studies. A simplified recombinant adenoviral system, AdEasy, will be applied to generate recombinant adenoviruses for the expression and knockdown of the gene of interest. We discuss the construction of vectors for gene expression and siRNAs, the generation of adenoviral recombinants, and the production of high-titer adenovirus. Furthermore, a procedure for injection of the adenovirus using a stereotaxic apparatus is discussed.