The nucleotide sequence of the adenovirus serotype 3 E2A early promoter has been determined. In contrast to Ad2, the Ad3 E2A early promoter possessed only one TATA-like box and one nuclear transcription factor E2F binding site and lacked the silencer sequences; however, as in Ad2, the ATF binding site was present. Moreover, the Ad3 E2A promoter harbored a protein binding sequence recognized by the SP1 factor. By transient expression analysis in HeLa cells, we demonstrated that the E1A gene products of Ad3 and Ad2 stimulated Ad3 E2A transcription. In competition experiments, the Ad3 E2A promoter was used in preference to the Ad2 E2A promoter.