Sp1 is a ubiquitous activator of numerous TATA-containing and TATA-less promoters within the human genome. This transcription factor is distinct from several other mammalian activators because it cannot stimulate transcription of reporter genes when ectopically expressed in Saccharomyces cerevisiae . Here we report that in cultured cells from Drosophila melanogaster human Sp1 efficiently activates transcription from synthetic promoters containing TATA boxes, but not from promoters that contain an initiator instead of a TATA box. The inability of Sp1 to activate initiator-mediated transcription did not result from inactivity of the consensus initiator element used for the experiments, as other initiator functions were conserved in Drosophila cells. Interestingly, a difference between the Drosophila and human TFIID complexes was found to be responsible for the selective inability of Sp1 to activate initiator-mediated transcription in Drosophila; in a complementation assay with a TFIID-depleted HeLa cell extract both the Drosophila and human TFIID complexes supported TATA-mediated transcription, but only the human complex supported initiator-mediated transcription. These results suggest that a species-specific interaction is required for activation of TATA-less promoters by Sp1, revealing a difference in transcriptional activation mechanisms between vertebrates and invertebrates.