E2F is a cellular, sequence-specific DNA-binding factor that binds to pairs of sites that occur upstream of the E1A and E2 early mRNA cap sites. During adenovirus infection, there is induction of a form of E2F that binds cooperatively to the pair of sites in the E2 control region. Production of the infection-specific E2F activity is dependent on early region 4 (E4), as extracts of cells infected with a mutant that lacks E4 did not contain this activity. Instead, two new forms of E2F were seen with the E4 mutant. Infection with mutant viruses unable to make E1A gene products produced the wild-type infection-specific E2F activity after a delay. Mutations in the E1B-55 kD-, E1B-21 kD-, E2-72 kD-, and E3-coding regions had no effect on production of infection-specific E2F. Analysis of cell lines confirmed the results obtained with mutant viruses. Cells that expressed E1A but not E4 genes (e.g., 293 cells) did not contain infection-specific E2F. Cell lines that expressed the E4 gene contained the activity. These observations demonstrate that E4 participates in the infection-induced change in E2F-binding activity. The data are consistent with E1A playing an indirect role in the process by mediating the efficient expression of E4 gene products which, in turn, induce the alteration in E2F activity.