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. ElA proteins can dissociate these complexes, releasing free E2F. This activity of ElA is independent of conserved domain 3 but is dependent on conserved domain 2 sequence. The ElAmediated 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 ElA in mediating a dissociation of transcription factor complexes, allowing new interactions to form and thus changing the transcriptional specificity.