Nuclear factors NFI and NFIII are involved in transcription of the E1A oncogene of adenovirus 12. In addition, the E-box binding transcription factor ESF-1 was found to activate basal transcription from the proximal transcription start site TS2. Deletion of a region upstream from the distal start site TS1 was reported to abolish E1A transcription completely. Two motifs for transcription factors, one for members of the E2F family and one that was related to ATF motifs in the HTLV-1 LTR, are localized in this region. We examined the binding of nuclear proteins to these motifs and studied their role in (auto)activation of Ad12 E1A transcription from TS1. We found several cell type specific DNA-protein complexes in Electrophoretic Mobility Shift Assays (EMSA). For HeLa, 293, U937, and A549 cells, participation of E2F-1, DP-1, cyclin A, and RB was involved in formation of some complexes only, assuming participation of factors different from E2F-1 or DP-1 in others. One main and 2-3 minor specific complexes appeared in EMSA when the ATF-motif was examined. Partial cross-competitions occurred in competition experiments between the neighbouring E2F and ATF-motifs, suggesting cofactors or bridging proteins in formation or stabilization of some complexes. Transcription from TS1 mediated by these motifs was analysed using a CAT-reporter system, where neither the ATF- nor the E2F-motif alone imparted striking activation of transcription. In contrast, considerable and synergistic activation was observed when both sites were present in the CAT-construct. E1A autoactivation mediated by these sites was about twofold compared with a ninefold activation described for the complete E1A promoter.