Adenoviral vectors have been widely used as efficient gene delivery vehicles for gene therapy and vaccine development, as well as gene function studies. The recently developed AdEasy system is a simple and efficient method for rapid generation of recombinant adenoviruses. Unlike traditional adenoviral vectors, the viral backbone described here is supplied as a supercoiled plasmid rather than as a linear viral DNA, facilitating backbone amplification. The recombination step is performed in E. coli rather than in mammalian cells, taking advantage of the high efficiency of homologous recombination in bacteria. This unit includes the complete set of protocols needed to generate recombinant adenoviruses using the AdEasy system.