The adenovirus large E1A protein is a potent activator of transcription. We use several different experimental approaches to demonstrate that the large E1A protein binds specifically and stably to the TATA box-binding factor (TFIID), the general polymerase II transcription factor that initiates assembly of transcription complexes. Sedimentation velocity centrifugation revealed that TFIID and E1A form a heterodimer in vitro. We demonstrate that the activation domain of E1A (conserved region 3) binds to TFIID. E1A interacts with a 51 residue region from the conserved C-terminal domain of TFIID that includes a repeat of basic residues between the homologous direct repeats of TFIID. Analysis of TFIID binding by various E1A mutants indicates that TFIID binding is necessary, although not sufficient, for E1A transactivation.