This chapter describes a novel strategy that simplifies the generation and production of adenovirus type 5 (Ad5) mutants carrying defined mutations in early transcription units 1 (E1) and 4 (E4). The strategy involves three recombinant plasmids containing E1 (pE1-1235), E4 (pE4-1155), or the wild-type genome that lacks a portion of E3 (pH5pg4100). To generate recombinant viruses, mutations are first introduced into pE1- and/or pE4-transfer plasmids by site-directed mutagenesis. The mutagenized constructs are then ligated into plasmid pH5pg4100 containing the Ad backbone by direct cloning. Infectious viral DNAs are released from the recombinant plasmids by PacI-digestion and transfected into the complementing cell lines 293 or W162, and viral progeny are isolated and amplified. The advantages of this strategy are multiple: all cloning steps are carried out in Escherichia coli, and any genetic region of the viral E1 and/or E4 transcription units can be specifically modified or deleted. Moreover, foreign genes can be introduced into the E1 and/or E4 regions, and expression of viral or therapeutic genes can be controlled by cell-type specific and/or inducible promoters.