Stimulation of in vitro transcription mediated by the upstream element of the adenovirus-2 major late promoter (Ad2MLP) involves its recognition by a specific trans-acting factor present in a HeLa whole-cell extract. DNase I footprinting and dimethylsulfate methylation protection experiments were used to determine, at the nucleotide level, upstream sequences which interact with this transcription factor. The ability of upstream element mutants to bind the transcription factor correlates directly with the efficiency of transcription from the corresponding Ad2ML promoters in vivo and in vitro. Competition footprinting experiments show that the transcription factor, which binds to the upstream element of the Ad2MLP, can also interact, but with a lower affinity, with the upstream elements of the Ad2E2a and rabbit beta-globin promoters, both of which display some sequence homology to the 'interacting' region of the Ad2MLP upstream element. The transcription factor does not, however, interact with the upstream elements of either the Ad2E2L, Ad5E3, SV40 early, herpes virus thymidine kinase or chicken conalbumin promoters.