In a variety of Drosophila TATA-less promoters, transcription is directed by initiator (Inr) sequences which are faithfully and efficiently recognized only when flanked 3' by the downstream promoter element or DPE. This motif, which is conserved at approximately 30 bp from the RNA start site, is viewed as a downstream counterpart to the TATA box, and is recognized by the general transcription factor TFIID. By transient expression assays in human embryonic kidney (HEK293)-cells, we show that DE1 (distal element 1), a DNA motif located at residues +23 to +29, sustains faithful Inr-dependent transcription as efficiently as the DPE. Transcription significantly increased when DE1 and DPE sequences were adjacently placed on the same template. Results emerging from in vivo RNA analyses matched Electrophoretic Mobility Shift Asssays (EMSA) data. In agarose-EMSAs, retarded DNA-protein complexes resulting from the interaction of human holo-TFIID with either Inr+/DPE+ or Inr+/DE1+ promoters were formed at comparable levels, while binding of TFIID to both DE1 and DPE motifs were two fold increased. The strict requirement for spacing between the Inr and DPE was not observed for DE1, as locating the motif 4 bp away from the +1 site did not impair transcriptional enhancement. DE1 sequences may be common to many promoters, and be underlooked because of their poor sequence homology.