Early region 1 (E1) of the human adenoviruses has many intriguing properties which have prompted numerous mutational studies to help delineate and characterize the domains responsible for these functions. In mutational analyses being done currently, the E1 region is usually cloned into a bacterial plasmid where it is mutated and then the altered E1 sequences are "rescued" back into infectious virus. The most frequently used rescue procedures are somewhat tedious, requiring the purification and fractionation of linear viral DNA or DNA fragments, and often involve the screening of numerous plaque isolates. Several observations we have made recently on the properties of adenovirus DNA in infected cells and on infectious plasmids in transfected cells led us to design a new approach for rescuing E1 mutations into infectious viral genomes. We constructed a plasmid, pJM17, containing the entire Ad5 DNA molecule, with an insert in the E1 region that exceeds the packaging constraints of the adenovirus capsid. Following transfection of pJM17 into 293 cells the plasmid DNA is able to replicate but cannot be packaged into infectious virions. In contrast cotransfection of 293 cells with pJM17 plus an E1-containing plasmid carrying mutated sequences produces recombinant virions at high efficiencies. Neither plasmid needs to be linearized prior to contransfection. The technique eliminates the need to purify and manipulate infectious virion DNA and since no unique restriction sites are needed, both E1A and E1B mutants' as well as foreign gene inserts in the E1 region can be easily rescued into virus.