Adenovirus infection activates the E2F transcription factor, in part through the formation of a heteromeric protein complex involving a 19 kd E4 gene product that then allows cooperative and stable promoter binding. We now find that cellular factors are complexed to E2F in extracts of several uninfected cell lines. E1A proteins can dissociate these complexes, releasing free E2F. This activity of E1A is independent of conserved domain 3 but is dependent on conserved domain 2 sequence. The E1A-mediated dissociation of the complexes allows the E4 protein to interact with E2F, generating a stable DNA-protein complex with the E2 promoter and a stimulation of transcription. These experiments demonstrate a function for E1A in mediating a dissociation of transcription factor complexes, allowing new interactions to form and thus changing the transcriptional specificity.