We describe the identification and characterization of a conserved downstream basal promoter element that is present in a subset of Drosophila TATA-box-deficient (TATA-less) promoters by using purified, epitope-tagged TFIID complex (eTFIID) from embryos of transgenic Drosophila. DNase I footprinting of the binding of eTFIID to TATA-less promoters revealed that the factor protected a region that extended from the initiation site sequence (about +1) to approximately 35 nucleotides downstream of the RNA start site. In contrast, there was no apparent upstream DNase I protection or hypersensitivity induced by eTFIID in the -25 to -30 region at which TATA motifs are typically located. Further studies revealed a conserved sequence motif, (A/G)G(A/T)CGTG, termed the downstream promoter element (DPE), which is located approximately 30 nucleotides downstream of the RNA start site of many TATA-less promoters. DNase I footprinting and in vitro transcription experiments revealed that a DPE in its normal downstream location is necessary for transcription of DPE-containing TATA-less promoters and can compensate for the disruption of an upstream TATA box of a TATA-containing promoter. Moreover, a systematic mutational analysis of DNA sequences that encompass the DPE confirmed the importance of the consensus DPE sequence motif for basal transcription and further supports the postulate that the DPE is a distinct, downstream basal promoter element. These results suggest that the DPE acts in conjunction with the initiation site sequence to provide a binding site for TFIID in the absence of a TATA box to mediate transcription of TATA-less promoters.