Two binding sites for EivF, a factor involved in transcription from the adenovirus early promoter iv (Eiv), were mapped within the adenovirus inverted terminal repeats (ITR). Consistent with the observation that EivF was required to initiate transcription from the Eiv promoter and with the demonstration that two EivF binding sites were present in the ITR, we show that the inverted terminal repeat region was able to promote transcription from the CAP site of the Eiv promoter in vitro and in an EIa-dependent fashion in vivo. The minimum sequence within the ITR capable of directing EIa-dependent transcription consists of forty nine nucleotides comprising two EivF binding sites and at least one Sp1 binding site. This 49-base pair fragment possesses the characteristics of an enhancer which is induced by EIa. The enhancer is active in HeLa cell nuclear extracts. Transcription directed by the ITR required EivF and the general transcription factors. The addition of purified Sp1 factor specifically stimulated transcription which correlates with the presence of Sp1 binding sites between the two EivF recognition sites.