Adenoviruses (Ads) possess several features that make them attractive mammalian gene transfer vectors. They can efficiently infect a wide variety of quiescent and proliferating cell types from various species to direct high level viral gene expression, their 36 kb double-stranded DNA genome can be manipulated with relative ease by conventional molecular biology techniques, and they can be readily propagated and purified to yield high titer preparations of very stable virus. Consequently, Ads have been extensively used as vectors for recombinant vaccines, for high-level protein production in cultured cells and for gene therapy where high-level, transient transgene expression is desired. This chapter describes in detail methods for the production and characterization of E1-deleted, first generation adenoviral vectors.