The binding of E2F to the adenovirus (Ad) E2a promoter is stimulated by the Ad E4-6/7 protein. E2F DNA binding activity is composed of a heterodimer of related but distinct proteins of the E2F-1 and DP-1 families. The E4-6/7 protein induces the cooperative and stable binding of E2F to an inverted repeat binding site in the E2a promoter apparently by providing a dimerization interface to two adjacent E2F heterodimers. The product of the retinoblastoma gene product (Rb) represses the transcriptional activity of E2F by direct protein-protein interaction. In this report, we have examined the regions of E2F-1 and DP-1 that are required for the induction of cooperative E2F binding to the E2a promoter by the E4-6/7 protein. Our results demonstrate that an internal segment of E2F-1, that is conserved among members of the E2F family, is required for functional interaction with the E4-6/7 product. Consistent with this observation, other members of the E2F family (E2F-2 and E2F-3) productively interact with E4-6/7. DP-1 also is necessary for stable interaction with E4-6/7 and an internal segment of DP-1 is required that is positioned in a location similar to that of the conserved E2F-1 domain. Interestingly, the binding of E4-6/7 and the binding of Rb to E2F are mutually exclusive, and our results show that the same internal segments of E2F-1 and DP-1 that are required for E4-6/7 binding are also required for stable interaction with Rb. These results suggest that the Ad E4-6/7 protein mimics Rb in part for the protein interaction requirements for E2F binding, although with different functional consequences. While Rb binding represses E2F activity, the E4-6/7 protein stimulates transactivation of the Ad E2a promoter.